T-cell modulatory multimeric polypeptides and methods of use thereof

ABSTRACT

The present disclosure provides variant immunomodulatory polypeptides, and fusion polypeptides comprising the variant immunomodulatory peptides. The present disclosure provides T-cell modulatory multimeric polypeptides, and compositions comprising same, where the T-cell modulatory multimeric polypeptides comprise a variant immunomodulatory polypeptide of the present disclosure. The present disclosure provides nucleic acids comprising nucleotide sequences encoding the T-cell modulatory multimeric polypeptides, and host cells comprising the nucleic acids. The present disclosure provides methods of modulating the activity of a T cell; the methods comprise contacting the T cell with a T-cell modulatory multimeric polypeptide of the present disclosure.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/438,272, filed Dec. 22, 2016, U.S. Provisional PatentApplication No. 62/470,774, filed Mar. 13, 2017. U.S. Provisional PatentApplication No. 62/555,435, filed Sep. 7, 2017, and U.S. ProvisionalPatent Application No. 62/582,132, filed Nov. 6, 2017, each of whichapplications is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“CUEB-107WO_SEQ_LISTING_171133_ST25.txt” created on Nov. 14, 2017 andhaving a size of 153 KB. The contents of the text file are incorporatedby reference herein in their entirety.

INTRODUCTION

An adaptive immune response involves the engagement of the T cellreceptor (TCR), present on the surface of a T cell, with a small peptideantigen non-covalently presented on the surface of an antigen presentingcell (APC) by a major histocompatibility complex (MHC; also referred toin humans as a human leukocyte antigen (HLA) complex). This engagementrepresents the immune system's targeting mechanism and is a requisitemolecular interaction for T cell modulation (activation or inhibition)and effector function. Following epitope-specific cell targeting, thetargeted T cells are activated through engagement of costimulatoryproteins found on the APC with counterpart costimulatory proteins the Tcells. Both signals—epitope/TCR binding and engagement of APCcostimulatory proteins with T cell costimulatory proteins—are requiredto drive T cell specificity and activation or inhibition. The TCR isspecific for a given epitope; however, the costimulatory protein is notepitope specific and instead is generally expressed on all T cells or onlarge T cell subsets.

SUMMARY

The present disclosure provides variant immunomodulatory polypeptides,and fusion polypeptides comprising the variant immunomodulatorypeptides. The present disclosure provides T-cell modulatory multimericpolypeptides, and compositions comprising same, where the T-cellmodulatory multimeric polypeptides comprise a variant immunomodulatorypolypeptide of the present disclosure. The present disclosure providesnucleic acids comprising nucleotide sequences encoding the T-cellmodulatory multimeric polypeptides, and host cells comprising thenucleic acids. The present disclosure provides methods of modulating theactivity of a T cell; the methods comprise contacting the T cell with aT-cell modulatory multimeric polypeptide of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D schematically depict various embodiments of a T-cellmodulatory multimeric polypeptide of the present disclosure. In theseembodiments, disulfide bonds are formed between MHC (e.g., HLA)polypeptides present in separate polypeptides.

FIG. 2A-2Q provide an amino acid sequence of wild-type human IL-2 (FIG.2A); and amino acid sequences of variant IL-2 polypeptides (FIG. 2B-2Q).

FIG. 3A-3C provide amino acid sequences of IL-2 receptor alpha chain(FIG. 3A), beta chain (FIG. 3B), and gamma chain (FIG. 3C).

FIG. 4A-4C provide amino acid sequences of immunoglobulin Fcpolypeptides.

FIG. 5A-5C provide amino acid sequences of human leukocyte antigen (HLA)Class I heavy chain polypeptides. Signal sequences are underlined.

FIG. 6 provides a multiple amino acid sequence alignment of beta-2microglobulin (β2M) precursors (i.e., including the leader sequence)from Homo sapiens (NP_004039.1; SEQ ID NO:95), Pan troglodytes(NP_001009066.1; SEQ ID NO:96), Macaca mulatta (NP_001040602.1; SEQ IDNO:97), Bos Taurus (NP_776318.1; SEQ ID NO:98) and Mus musculus(NP_033865.2; SEQ ID NO:99). Amino acids 1-20 are a signal peptide.

FIG. 7A-7B depict production of IL-2/synTacs (“Cue-IL-2-a” andCue-IL-2-b”) of the present disclosure following transient transfection.FIG. 7A depicts unpurified yields; FIG. 7B depicts purified product.

FIG. 8A-8B depict production of IL-2/synTacs of the present disclosure,in which the IL-2 polypeptide is present on the light chain (thepolypeptide chain with the light chain (e.g., β2M) of an MHC Class Imolecule) or on the heavy chain (the polypeptide chain with the heavychain of an MHC Class I molecule).

FIG. 9 depicts the expression level of IL-2/syn-Tacs, in which the IL-2is wild-type (wt), or comprises various combinations of F42A, D20K,Q126A, E15A, Y45A, and H16A.

FIG. 10 depicts expression of IL-2/synTacs of the present disclosure, inwhich the IL-2 is present in one copy (1×), two copies (2×) or threecopies (3×) in the synTac.

FIG. 11 depicts in vitro stimulation of antigen-specific CD8⁻ T cellsand non-specific CD8⁺ T cells by an IL-2/synTac of the presentdisclosure, where the IL-2 variant comprising F42A and H16Asubstitutions is present in the synTac in two copies.

FIG. 12 depicts IL-2/synTac binding to specific (lymphocyticchoriomeningitis virus; LCMV) or non-specific (OT1; recognizingovalbumin) CD8⁺ T cells.

FIG. 13 depicts IL-2/synTac-mediated signaling in antigen-specific(LCMV) or non-specific (BL6) CD8⁺ T cells.

FIG. 14A-14F depict the percent phospho-signal transducer and activatorof transcription 5 (pSTATS)-positive cells following stimulation of CD8⁺antigen-specific (LCMV) or non-specific (BL6) cells with IL-2/synTacs ofthe present disclosure at various IL-2/synTac concentrations.

FIG. 15 depicts in vivo activity of an IL-2/synTac of the presentdisclosure. The left panel depicts the fold change in the number ofantigen-specific CD8⁺ T cells following administration of phosphatebuffered saline (PBS), recombinant IL-2 (rIL-2), or an IL-2/synTac ofthe present disclosure. The right panel depicts antigen-specific andnon-antigen-specific responses following administration of PBS, rIL-2,or an IL-2/synTac of the present disclosure.

FIG. 16A-16B depict dose escalation (FIG. 16A) and route ofadministration (FIG. 16B) effects.

FIG. 17A-17B depict the effect of IL-2 copy number on in vivo efficacyagainst a tumor.

FIG. 18 depicts the serum half-life of an IL-2/synTac of the presentdisclosure, following intraperitoneal administration of the IL-2/synTacin an amount of 10 mg/kg.

FIG. 19 depicts stability of an IL-2/synTac of the present disclosure 2hours following intraperitoneal administration of the IL-2/synTac in anamount of 10 mg/kg.

FIG. 20 depicts size exclusion chromatography data on an IL-2/synTac ofthe present disclosure after keeping the IL-2/synTac at 4° C., or 37° C.for 5 days.

FIG. 21 provides an amino acid sequence of a heavy chain of anIL-2/synTac of the present disclosure, with a leader peptide, where theIL-2/synTac heavy chain comprises an IgG1 Fc with an N297A substitution.

FIG. 22 provides an amino acid sequence of a heavy chain of anIL-2/synTac of the present disclosure, without a leader peptide, wherethe IL-2/synTac heavy chain comprises an IgG1 Fc with an N297Asubstitution.

FIG. 23A-23B provide a nucleotide sequence (FIG. 23A) encoding theIL-2/synTac heavy chain depicted in FIG. 21; and a key (FIG. 23B) to thesequence.

FIG. 24 provides an amino acid sequence of a heavy chain of anIL-2/synTac of the present disclosure, with a leader peptide, where theIL-2/synTac heavy chain comprises an IgG1 Fc with L234A and L235Asubstitutions.

FIG. 25 provides an amino acid sequence of a heavy chain of anIL-2/synTac of the present disclosure, without a leader peptide, wherethe IL-2/synTac heavy chain comprises an IgG1 Fc with L234A and L235Asubstitutions.

FIG. 26A-26B provide a nucleotide sequence (FIG. 26A) encoding theIL-2/synTac heavy chain depicted in FIG. 24; and a key (FIG. 26B) to thesequence.

FIG. 27 provides an amino acid sequence of a heavy chain of anIL-2/synTac of the present disclosure, with a leader peptide, where theIL-2/synTac heavy chain comprises an IgG1 Fc with L234F, L235E, andV331S substitutions.

FIG. 28 provides an amino acid sequence of a heavy chain of anIL-2/synTac of the present disclosure, without a leader peptide, wherethe IL-2/synTac heavy chain comprises an IgG1 Fc with L234F, L235E, andP331S substitutions.

FIG. 29A-29B provide a nucleotide sequence (FIG. 29A) encoding theIL-2/synTac heavy chain depicted in FIG. 27; and a key (FIG. 29B) to thesequence.

FIG. 30 provides an amino acid sequence of a light chain of anIL-2/synTac of the present disclosure, with a leader peptide, where theIL-2/synTac light chain comprises a human papilloma virus (HPV) E7epitope.

FIG. 31 provides an amino acid sequence of a light chain of anIL-2/synTac of the present disclosure, without a leader peptide, wherethe IL-2/synTac light chain comprises an HPV E7 epitope.

FIG. 32 provides a nucleotide sequence encoding the IL-2/synTac lightchain depicted in FIG. 30.

FIG. 33A-33D provide amino acid sequences of a wild-type human IgG1 Fc(FIG. 33A), an IgG1 Fc with L234F, L235E, and P331S substitutions (FIG.33B), an IgG1 Fc with an N297A substitution (FIG. 33C), and an IgG1 Fcwith L234A and L235A substitutions (FIG. 33D).

FIG. 34A-34C provide amino acid sequence of a β2-microglobulin (R12C)polypeptide (FIG. 34A), a variant IL-2 (H16A; F42A) polypeptide (FIG.34B), and a Class I MHC-H chain A0201 (Y84A; A236C) (FIG. 34C).

FIG. 35 depicts IL-2/synTac-mediated expansion of human CMV-specificCD8⁺ T cells.

FIG. 36 provides expression data and receptor binding data for synTacswith variant IL-2 polypeptides.

FIG. 37 depicts binding of an IL-2/synTac to primary human HPV16 E7(11-20)-specific CD8⁺ T cells, as detected by flow cytometry.

FIG. 38 depicts the effect of binding of the variant IL-2/synTac toprimary human HPV16 E7 (11-20)-specific CD8⁺ T cells on phosphorylationof SLP76.

FIG. 39 depicts the effect of binding of the variant IL-2/synTac toprimary human HPV16 E7 (11-20)-specific T cells on production of CD25,granzyme B, and CD107α.

FIG. 40 depicts the effect of binding of the variant IL-2/synTac toprimary human HPV16 E7 (11-20)-specific CD8⁺ T cells on production ofIFN-γ.

DEFINITIONS

The terms “polynucleotide” and “nucleic acid.” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same, and inthe same relative position, when comparing the two sequences. Sequenceidentity can be determined in a number of different ways. To determinesequence identity, sequences can be aligned using various convenientmethods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT,etc.), available over the world wide web at sites includingncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/,ebi.ac.uk/Tools/msa/muscle/, mafft.ebre.jp/alignment/software/. See,e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains. For example, a group of amino acids having aliphatic sidechains consists of glycine, alanine, valine, leucine, and isoleucine; agroup of amino acids having aliphatic-hydroxyl side chains consists ofserine and threonine; a group of amino acids having amide containingside chains consisting of asparagine and glutamine; a group of aminoacids having aromatic side chains consists of phenylalanine, tyrosine,and tryptophan; a group of amino acids having basic side chains consistsof lysine, arginine, and histidine; a group of amino acids having acidicside chains consists of glutamate and aspartate; and a group of aminoacids having sulfur containing side chains consists of cysteine andmethionine. Exemplary conservative amino acid substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine-glycine, and asparagine-glutamine.

“Binding” as used herein (e.g. with reference to binding of a T-cellmodulatory multimeric polypeptide of the present disclosure to apolypeptide (e.g., a T-cell receptor) on a T cell) refers to anon-covalent interaction between. Binding interactions are generallycharacterized by a dissociation constant (K_(D)) of less than 10⁻⁶ M,less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻¹⁰ M,less than 10⁻¹¹ M, less than 10⁻¹² M, less than 10⁻¹³ M, less than 10⁻¹⁴M, or less than 10⁻¹⁵ M. “Affinity” refers to the strength of binding,increased binding affinity being correlated with a lower K_(D).

The term “immunological synapse” or “immune synapse” as used hereingenerally refers to the natural interface between two interacting immunecells of an adaptive immune response including, e.g., the interfacebetween an antigen-presenting cell (APC) or target cell and an effectorcell, e.g., a lymphocyte, an effector T cell, a natural killer cell, andthe like. An immunological synapse between an APC and a T cell isgenerally initiated by the interaction of a T cell antigen receptor andmajor histocompatibility complex molecules, e.g., as described inBromley et al., Annu Rev Immunol. 2001; 19:375-96; the disclosure ofwhich is incorporated herein by reference in its entirety.

“T cell” includes all types of immune cells expressing CD3, includingT-helper cells (CD4⁺ cells), cytotoxic T-cells (CD8⁺ cells),T-regulatory cells (Treg), and NK-T cells.

“Co-stimulatory polypeptide,” as the term is used herein, includes apolypeptide on an antigen presenting cell (APC) (e.g., a dendritic cell,a B cell, and the like) that specifically binds a cognate co-stimulatorypolypeptide on a T cell, thereby providing a signal which, in additionto the primary signal provided by, for instance, binding of a TCR/CD3complex with a major histocompatibility complex (MHC) polypeptide loadedwith peptide, mediates a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like. Aco-stimulatory ligand can include, but is not limited to, CD7, B7-1(CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL),inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule(ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxinbeta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody thatbinds Toll ligand receptor and a ligand that specifically binds withB7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibodythat specifically binds with a co-stimulatory molecule present on a Tcell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40,PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

A “modulatory domain” (“MOD”) of a T-cell modulatory multimericpolypeptide of the present disclosure comprises a co-stimulatorypolypeptide, e.g., an IL-2 polypeptide, such as a variant IL-2polypeptide of the present disclosure.

“Heterologous,” as used herein, means a nucleotide or polypeptide thatis not found in the native nucleic acid or protein, respectively.

“Recombinant,” as used herein, means that a particular nucleic acid (DNAor RNA) is the product of various combinations of cloning, restriction,polymerase chain reaction (PCR) and/or ligation steps resulting in aconstruct having a structural coding or non-coding sequencedistinguishable from endogenous nucleic acids found in natural systems.DNA sequences encoding polypeptides can be assembled from cDNA fragmentsor from a series of synthetic oligonuclotides, to provide a syntheticnucleic acid which is capable of being expressed from a recombinanttranscriptional unit contained in a cell or in a cell-free transcriptionand translation system.

The terms “recombinant expression vector,” or “DNA construct” are usedinterchangeably herein to refer to a DNA molecule comprising a vectorand one insert. Recombinant expression vectors are usually generated forthe purpose of expressing and/or propagating the insert(s), or for theconstruction of other recombinant nucleotide sequences. The insert(s)may or may not be operably linked to a promoter sequence and may or maynot be operably linked to DNA regulatory sequences.

A cell has been “genetically modified” or “transformed” or “transfected”by exogenous DNA, e.g., a recombinant expression vector, when such DNAhas been introduced inside the cell. The presence of the exogenous DNAresults in permanent or transient genetic change. The transforming DNAmay or may not be integrated (covalently linked) into the genome of thecell. In prokaryotes, yeast, and mammalian cells, for example, thetransforming DNA may be maintained on an episomal element such as aplasmid. With respect to eukaryotic cells, a stably transformed cell isone in which the transforming DNA has become integrated into achromosome so that it is inherited by daughter cells through chromosomereplication.

A “host cell.” as used herein, denotes an in vivo or in vitro eukaryoticcell or a cell from a multicellular organism (e.g., a cell line)cultured as a unicellular entity, which eukaryotic cells can be, or havebeen, used as recipients for a nucleic acid (e.g., an expression vectorthat comprises a nucleotide sequence encoding a multimeric polypeptideof the present disclosure), and include the progeny of the original cellwhich has been genetically modified by the nucleic acid. It isunderstood that the progeny of a single cell may not necessarily becompletely identical in morphology or in genomic or total DNA complementas the original parent, due to natural, accidental, or deliberatemutation. A “recombinant host cell” (also referred to as a “geneticallymodified host cell”) is a host cell into which has been introduced aheterologous nucleic acid, e.g., an expression vector. For example, agenetically modified eukaryotic host cell is genetically modified byvirtue of introduction into a suitable eukaryotic host cell aheterologous nucleic acid, e.g., an exogenous nucleic acid that isforeign to the eukaryotic host cell, or a recombinant nucleic acid thatis not normally found in the eukaryotic host cell.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease or symptom in a mammal, and includes:(a) preventing the disease or symptom from occurring in a subject whichmay be predisposed to acquiring the disease or symptom but has not yetbeen diagnosed as having it; (b) inhibiting the disease or symptom,i.e., arresting its development; or (c) relieving the disease, i.e.,causing regression of the disease. The therapeutic agent may beadministered before, during or after the onset of disease or injury. Thetreatment of ongoing disease, where the treatment stabilizes or reducesthe undesirable clinical symptoms of the patient, is of particularinterest. Such treatment is desirably performed prior to complete lossof function in the affected tissues. The subject therapy will desirablybe administered during the symptomatic stage of the disease, and in somecases after the symptomatic stage of the disease.

The terms “individual,” “subject,” “host,” and “patient,” are usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired. Mammals include, e.g.,humans, non-human primates, rodents (e.g., rats; mice), lagomorphs(e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, andthe like), etc.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications artcited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an.” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “avariant IL-2 polypeptide” includes a plurality of such polypeptides andreference to “the Class I HLA heavy chain polypeptide” includesreference to one or more Class I HLA heavy chain polypeptides andequivalents thereof known to those skilled in the art, and so forth. Itis further noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides variant immunomodulatory polypeptides,and fusion polypeptides comprising the variant immunomodulatorypeptides. The present disclosure provides T-cell modulatory multimericpolypeptides, and compositions comprising same, where the T-cellmodulatory multimeric polypeptides comprise a variant immunomodulatorypolypeptide of the present disclosure. The present disclosure providesnucleic acids comprising nucleotide sequences encoding the T-cellmodulatory multimeric polypeptides, and host cells comprising thenucleic acids. The present disclosure provides methods of modulating theactivity of a T cell; the methods comprise contacting the T cell with aT-cell modulatory multimeric polypeptide of the present disclosure.

In embodiments described herein, a multimeric polypeptide functions as asurrogate APC, and mimics the adaptive immune response. The multimericpolypeptide does so by engaging a TCR present on the surface of a T cellwith an epitope-presenting peptide complexed with an MHC present in themultimeric polypeptide. This engagement provides the multimericpolypeptide with the ability to achieve epitope-specific cell targeting.In embodiments described herein, the multimeric polypeptide alsopossesses at least one immunomodulatory protein (also referred to hereinas a “modulatory domain” or “MOD”) that engages a counterpartcostimulatory protein (also referred to herein as an “immunomodulatorypolypeptide,” a “cognate immunomodulatory polypeptide,” or a “cognatecostimulatory protein.” and the like) on the T cell. Bothsignals—epitope/MHC binding to a TCR and immunomodulatory polypeptidebinding to a cognate costimulatory polypeptide—then drive both thedesired T cell specificity and either inhibition oractivation/proliferation. As further described herein, the at least oneimmunomodulatory protein may be a variant of a naturally occurringimmunomodulatory protein (e.g., naturally occurring IL-2), which variantexhibits a reduced affinity for its counterpart costimulatory protein onthe T cell (e.g., IL-2R) as compared to the affinity of the naturallyoccurring immunomodulatory protein for the counterpart costimulatoryprotein.

A T-cell modulatory multimeric polypeptide of the present disclosure isalso referred to as a “synTac polypeptide.” A synTac polypeptide of thepresent disclosure comprises a variant modulatory domain, where thevariant modulatory domain exhibits reduced binding affinity to animmunomodulatory polypeptide (a cognate costimulatory polypeptide, e.g.,a cognate costimulatory polypeptide on the surface of a T cell),compared to the affinity of a wild-type modulatory domain for theimmunomodulatory polypeptide. A synTac polypeptide of the presentdisclosure can modulate the activity of a target T-cell. A synTacpolypeptide of the present disclosure provides for enhanced target cellspecificity.

Variant Immunomodulatory Polypeptides

The present disclosure provides variant IL-2 modulatory polypeptides. Awild-type amino acid sequence of human IL-2 is provided in FIG. 2A. Awild-type amino acid sequence of a human IL-2 polypeptide can be asfollows: APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADETATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:1).

Wild-type IL-2 binds to an IL-2 receptor (IL-2R) on the surface of acell. An IL-2 receptor is in some cases a heterotrimeric polypeptidecomprising an alpha chain (IL-2Rα; also referred to as CD25), a betachain (IL-2Rβ; also referred to as CD122; and a gamma chain (IL-2Rγ;also referred to as CD132). Amino acid sequences of human IL-2Rα,IL-2Rβ, and IL-2Rγ are provided in FIG. 3A-3C.

In some cases, a variant IL-2 polypeptide of the present disclosureexhibits reduced binding affinity to IL-2R, compared to the bindingaffinity of an IL-2 polypeptide comprising the amino acid sequencedepicted in FIG. 2A for IL-2R. For example, in some cases, a variantIL-2 polypeptide of the present disclosure binds IL-2R with a bindingaffinity that is less than the binding affinity of an IL-2 polypeptidecomprising the amino acid sequence depicted in FIG. 2A for an IL-2Rpolypeptide comprising alpha, beta, and gamma chains comprising theamino acid sequences depicted in FIG. 3A-3C. For example, in some cases,a variant IL-2 polypeptide of the present disclosure binds IL-2R with abinding affinity that is at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50% less, at least 55% less, at least 60% less, atleast 65% less, at least 70% less, at least 75% less, at least 80% less,at least 85% less, at least 90% less, at least 95% less, or more than95% less, than the binding affinity of an IL-2 polypeptide comprisingthe amino acid sequence depicted in FIG. 2A for an IL-2R (e.g., an IL-2Rcomprising alpha, beta, and gamma chains comprising the amino acidsequences (mature forms) depicted in FIG. 3A-3C). In such cases, bindingaffinity is determined using the procedure described below.

In some cases, a variant IL-2 polypeptide of the present disclosure hasa binding affinity for IL-2R that is from 100 nM to 100 μM. As anotherexample, in some cases, a variant IL-2 polypeptide of the presentdisclosure has a binding affinity for IL-2R (e.g., an IL-2R comprisingalpha, beta, and gamma chains comprising the amino acid sequencesdepicted in FIG. 3A-3C) that is from about 100 nM to 150 nM, from about150 nM to about 200 nM, from about 200 nM to about 250 nM, from about250 nM to about 300 nM, from about 300 nM to about 350 nM, from about350 nM to about 400 nM, from about 400 nM to about 500 nM, from about500 nM to about 600 nM, from about 600 nM to about 700 nM, from about700 nM to about 800 nM, from about 800 nM to about 900 nM, from about900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μM toabout 10 μM, from about 10 μM to about 15 μM, from about 15 μM to about20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM,from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. Insuch cases, binding affinity is determined using the procedure describedbelow.

A variant IL-2 polypeptide of the present disclosure can have a singleamino acid substitution relative to a wild-type IL-2 polypeptide (e.g.,an IL-2 polypeptide comprising the amino acid sequence depicted in FIG.2A or as set forth in SEQ ID NO:1). In some cases, a variant IL-2polypeptide of the present disclosure has from 2 to 10 amino acidsubstitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL-2 polypeptide ofthe present disclosure has 2 amino acid substitutions relative to awild-type IL-2 polypeptide (e.g., an IL-2 polypeptide comprising theamino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO:1).In some cases, a variant IL-2 polypeptide of the present disclosure has3 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., an IL-2 polypeptide comprising the amino acid sequence depictedin FIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variantIL-2 polypeptide of the present disclosure has 4 amino acidsubstitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL-2 polypeptide ofthe present disclosure has 5 amino acid substitutions relative to awild-type IL-2 polypeptide (e.g., an IL-2 polypeptide comprising theamino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO:1).In some cases, a variant IL-2 polypeptide of the present disclosure has6 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., an IL-2 polypeptide comprising the amino acid sequence depictedin FIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variantIL-2 polypeptide of the present disclosure has 7 amino acidsubstitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL-2 polypeptide ofthe present disclosure has 8 amino acid substitutions relative to awild-type IL-2 polypeptide (e.g., an IL-2 polypeptide comprising theamino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO:1).In some cases, a variant IL-2 polypeptide of the present disclosure has9 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., an IL-2 polypeptide comprising the amino acid sequence depictedin FIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variantIL-2 polypeptide of the present disclosure has 10 amino acidsubstitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1).

A variant IL-2 polypeptide of the present disclosure can have a lengthof from 120 amino acids to 140 amino acids, e.g., from 120 amino acidsto 125 amino acids, from 125 amino acids to 130 amino acids, from 130amino acids to 135 amino acids, or from 135 amino acids to 140 aminoacids. In some cases, a variant IL-2 polypeptide of the presentdisclosure has a length of 133 amino acids.

E15 Substitution

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2D, where amino acid 15 is an amino acidother than a glutamic acid, e.g., where amino acid 15 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg,His, or Asp. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2D, where amino acid 15 isAla, Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2D, where aminoacid 15 is Ala. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2D, where amino acid 15 isGly. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2D, where amino acid 15 is Val. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2D, where amino acid 15 is Leu. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2D, where amino acid 15 is Ile. In some cases, the E15substitution variant IL-2 polypeptides described above have a bindingaffinity for IL2R that is from about 100 nM to 150 nM, from about 150 nMto about 200 nM, from about 200 nM to about 250 nM, from about 250 nM toabout 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 700 nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM,from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 μM. In somecases, such variant IL-2 polypeptides bind IL-2R with a binding affinitythat is at least 5%, 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% less,at least 55% less, at least 60% less, at least 65% less, at least 70%less, at least 75% less, at least 80% less, at least 85% less, at least90% less, at least 95% less, or more than 95% less, than the bindingaffinity of an IL-2 polypeptide comprising the amino acid sequencedepicted in FIG. 2A for an IL-2R (e.g., an IL-2R comprising alpha, beta,and gamma chains comprising the amino acid sequences (mature forms)depicted in FIG. 3A-3C). In some cases, such variant IL-2 polypeptidehas a length of 133 amino acids.

H16 Substitution

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2E, where amino acid 16 is an amino acidother than a histidine, e.g., where amino acid 16 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, orGlu. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2E, where amino acid 16 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2E, where amino acid 16 isAla. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2E, where amino acid 16 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Val. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Leu. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Ile. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Asn. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Asp. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Cys. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Gln. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Glu. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Met. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Phe. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Ser. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Thr. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Trp. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Tyr. In some cases, the H16substitution variant IL-2 polypeptides described above have a bindingaffinity for IL-2R that is from about 100 nM to 150 nM, from about 150nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nMto about 300 nμM, hum about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 700 nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM,from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 μM. In somecases, such variant IL-2 polypeptides bind IL-2R with a binding affinitythat is at least 5%, 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% less,at least 55% less, at least 60% less, at least 65% less, at least 70%less, at least 75% less, at least 80% less, at least 85% less, at least90% less, at least 95% less, or more than 95% less, than the bindingaffinity of an IL-2 polypeptide comprising the amino acid sequencedepicted in FIG. 2A for an IL-2R (e.g., an IL-2R comprising alpha, beta,and gamma chains comprising the amino acid sequences (mature forms)depicted in FIG. 3A-3C). In some cases, such variant IL-2 polypeptidehas a length of 133 amino acids.

D20 Substitution

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2C, where amino acid 20 is an amino acidother than an aspartic acid, e.g., where amino acid 20 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg,His, or Glu. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2C, where amino acid 20 isAla, Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2C, where aminoacid 20 is Ala. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2C, where amino acid 20 isGly. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2C, where amino acid 20 is Val. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2C, where amino acid 20 is Leu. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2C, where amino acid 20 is Ile. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2C, where amino acid 20 is Asn, Gln, Lys, Arg, or His.In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2C, where amino acid 20 is Lys. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2C, where amino acid 20 is Asn. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2C, where amino acid 20 is Gln. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2C, where amino acid 20 is His. In some cases, the D20substitution variant IL-2 polypeptides described above have a bindingaffinity for IL-2R that is from about 100 nM to 150 nM, from about 150nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nMto about 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 700 nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 tμM, to about 1 μM to about 5 M, from about 5 μM to about 10 μMfrom about 10 μM to about 15 μM, from about 15 μM to about 20 μM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 μM. In somecases, such variant IL-2 polypeptides bind IL-2R with a binding affinitythat is at least 5%, 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% less,at least 55% less, at least 60% less, at least 65% less, at least 70%less, at least 75% less, at least 80% less, at least 85% less, at least90% less, at least 95% less, or more than 95% less, than the bindingaffinity of an IL-2 polypeptide comprising the amino acid sequencedepicted in FIG. 2A for an IL-2R (e.g., an IL-2R comprising alpha, beta,and gamma chains comprising the amino acid sequences (mature forms)depicted in FIG. 3A-3C). In some cases, such variant IL-2 polypeptidehas a length of 133 amino acids.

F42 Substitution

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2B, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2B, where amino acid 42 isAla, Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2B, where aminoacid 42 is Ala. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2B, where amino acid 42 isGly. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2B, where amino acid 42 is Val. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2B, where amino acid 42 is Leu. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2B, where amino acid 42 is Ile. In some cases, the F42substitution variant IL-2 polypeptides described above have a bindingaffinity for IL2R that is from about 100 nM to 150 nM, from about 150 nMto about 200 nM, from about 200 nM to about 250 nM, from about 250 nM toabout 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 70×) nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM,from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 μM. In somecases, such variant IL-2 polypeptides bind IL2R with a binding affinitythat is at least 5%, 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% less,at least 55% less, at least 60% less, at least 65% less, at least 70%less, at least 75% less, at least 80% less, at least 85% less, at least90% less, at least 95% less, or more than 95% less, than the bindingaffinity of an IL-2 polypeptide comprising the amino acid sequencedepicted in FIG. 2A for an IL2R (e.g., an IL2R comprising alpha, beta,and gamma chains comprising the amino acid sequences (mature forms)depicted in FIG. 3A-3C). In some cases, such variant IL-2 polypeptidehas a length of 133 amino acids.

Y45 Substitution

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2F, where amino acid 45 is an amino acidother than a tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His. Asp, orGlu. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2F, where amino acid 45 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2F, where amino acid 45 isAla. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2F, where amino acid 45 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2F, where amino acid 45 is Val. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2F, where amino acid 45 is Leu. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2F, where amino acid 45 is Ile. In some cases, the Y45substitution variant IL-2 polypeptides described above have a bindingaffinity for IL2R that is from about 100 nM to 150 nM, from about 150 nMto about 200 nM, from about 200 nM to about 250 nM, from about 250 nM toabout 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 700 nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM,from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 μM. In somecases, such variant IL-2 polypeptides bind IL2R with a binding affinitythat is at least 5%, 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% less,at least 55% less, at least 60% less, at least 65% less, at least 70%less, at least 75% less, at least 80% less, at least 85% less, at least90% less, at least 95% less, or more than 95% less, than the bindingaffinity of an IL-2 polypeptide comprising the amino acid sequencedepicted in FIG. 2A for an IL2R (e.g., an IL2R comprising alpha, beta,and gamma chains comprising the amino acid sequences (mature forms)depicted in FIG. 3A-3C). In some cases, such variant IL-2 polypeptidehas a length of 133 amino acids.

Q126 Substitution

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2G, where amino acid 126 is an amino acidother than a glutamine, e.g., where amino acid 126 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His,Asp, or Glu. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2G, where amino acid 126 isAla, Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2G, where aminoacid 126 is Ala. In some cases, a valiant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2G, where aminoacid 126 is Gly. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2G, where aminoacid 126 is Val. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2G, where aminoacid 126 is Leu. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2G, where aminoacid 126 is Ile. In some cases, the Q126 substitution variant IL-2polypeptides described above have a binding affinity for IL2R that isfrom about 100 nM to 150 nM, from about 150 nM to about 200 nM, fromabout 200 nM to about 250 nM, from about 250 nM to about 300 nM, fromabout 300 nM to about 350 nM, from about 350 nM to about 400 nM, fromabout 400 nM to about 500 nM, from about 500 nM to about 600 nM, fromabout 600 nM to about 700 nM, from about 700 nM to about 800 nM, fromabout 800 nM to about 900 nM, from about 900 nM to about 1 μM, to about1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM toabout 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM,or from about 75 μM to about 100 μM. In some cases, such variant IL-2polypeptides hind IL2R with a binding affinity that is at least 5%, 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50% less, at least 55% less, at least60% less, at least 65% less, at least 70% less, at least 75% less, atleast 80% less, at least 85% less, at least 90% less, at least 95% less,or more than 95% less, than the binding affinity of an IL-2 polypeptidecomprising the amino acid sequence depicted in FIG. 2A for an IL2R(e.g., an IL2R comprising alpha, beta, and gamma chains comprising theamino acid sequences (mature forms) depicted in FIG. 3A-3C). In somecases, such variant IL-2 polypeptide has a length of 133 amino acids.

F42 and H16 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2H, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu; and where amino acid 16 is an amino acid other than ahistidine, e.g., where amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. Insome cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2H, where amino acid 42 is Ala, Gly, Val,Leu, or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. Insome cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2H, where amino acid 42 is Ala and aminoacid 16 is Ala. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2H, where amino acid 42 isAla and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptideof the present disclosure comprises an amino acid sequence having atleast 90%, at least 95%, at least 98%, or at least 99%, amino acidsequence identity to the amino acid sequence depicted in FIG. 2H, whereamino acid 42 is Val and amino acid 16 is Ala. In some cases, a variantIL-2 polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2H, where amino acid 42 is Leu, and amino acid 16 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2H, where amino acid 42 is Ile and amino acid 16 isAla. In some cases, the F42/H16 substitution variant IL-2 polypeptidesdescribed above have a binding affinity for IL2R that is from about 100nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM toabout 250 nM, from about 250 nM to about 300 nM, from about 300 nM toabout 350 nM, from about 350 nM to about 400 nM, from about 400 nM toabout 500 nM, from about 500 nM to about 600 nM, from about 600 nM toabout 700 nM, from about 700 nM to about 800 nM, from about 800 nM toabout 900 nM, from about 900 nM to about 1 μM, to about 1 μM to about 5μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM,from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, fromabout 25 μM to about 50 μM, from about 50 μM to about 75 μM, or fromabout 75 μM to about 100 μM. In some cases, such variant IL-2polypeptides bind IL2R with a binding affinity that is at least 5%, 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50% less, at least 55% less, at least60% less, at least 65% less, at least 70% less, at least 75% less, atleast 80% less, at least 85% less, at least 90% less, at least 95% less,or more than 95% less, than the binding affinity of an IL-2 polypeptidecomprising the amino acid sequence depicted in FIG. 2A for an IL2R(e.g., an IL2R comprising alpha, beta, and gamma chains comprising theamino acid sequences (mature forms) depicted in FIG. 3A-3C). In somecases, such variant IL-2 polypeptide has a length of 133 amino acids.

F42 and D20 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 21, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp. Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu; and where amino acid 20 is an amino acid other than anaspartic acid, e.g., where amino acid 20 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met. Asn, Gln, Lys, Arg, His, or Glu.In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2I, where amino acid 42 is Ala, Gly, Val,Leu, or Ile; and where amino acid 20 is Ala, Gly, Val, Leu, or Ile. Insome cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 21, where amino acid 42 is Ala, Gly, Val,Leu, or Ile; and where amino acid 20 is Asn, Gln, Lys, Arg, or His. Insome cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2I, where amino acid 42 is Ala and aminoacid 20 is Ala. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2I, where amino acid 42 isAla and amino acid 20 is Gly. In some cases, a variant IL-2 polypeptideof the present disclosure comprises an amino acid sequence having atleast 90%, at least 95%, at least 98%, or at least 99%, amino acidsequence identity to the amino acid sequence depicted in FIG. 2I, whereamino acid 42 is Val and amino acid 20 is Ala. In some cases, a variantIL-2 polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 21, where amino acid 42 is Leu, and amino acid 20 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2I, where amino acid 42 is Ile and amino acid 20 isAla. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2I, where amino acid 42 is Ala and aminoacid 20 is Asn. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2I, where amino acid 42 isAla and amino acid 20 is Gln. In some cases, a variant IL-2 polypeptideof the present disclosure comprises an amino acid sequence having atleast 90%, at least 95%, at least 98%, or at least 99%, amino acidsequence identity to the amino acid sequence depicted in FIG. 2I, whereamino acid 42 is Ala and amino acid 20 is Lys. In some cases, a variantIL-2 polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2I, where amino acid 42 is Ala and amino acid 20 is Arg. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2I, where amino acid 42 is Ala and amino acid 20 isHis. In some cases, the F42/D20 substitution variant IL-2 polypeptidesdescribed above have a binding affinity for IL2R that is from about 100nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM toabout 250 nM, from about 250 nM to about 300 nM, from about 300 nM toabout 350 nM, from about 350 nM to about 400 nM, from about 400 nM toabout 500 nM, from about 500 nM to about 600 nM, from about 600 nM toabout 700 nM, from about 700 nM to about 800 nM, from about 800 nM toabout 900 nM, from about 900 nM to about 1 μM, to about 1 μM to about 5μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM,from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, fromabout 25 μM to about 50 μM, from about 50 μM to about 75 μM, or fromabout 75 μM to about 100 μM. In some cases, such variant IL-2polypeptides bind IL2R with a binding affinity that is at least 5%, 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50% less, at least 55% less, at least60% less, at least 65% less, at least 70% less, at least 75% less, atleast 80% less, at least 85% less, at least 90% less, at least 95% less,or more than 95% less, than the binding affinity of an IL-2 polypeptidecomprising the amino acid sequence depicted in FIG. 2A for an IL2R(e.g., an IL2R comprising alpha, beta, and gamma chains comprising theamino acid sequences (mature forms) depicted in FIG. 3A-3C). In somecases, such variant IL-2 polypeptide has a length of 133 amino acids.

F42, D20, and E15 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90b, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2J, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu; where amino acid 20 is an amino acid other than an asparticacid, e.g., where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu; and whereamino acid 15 is an amino acid other than a glutamic acid, e.g., whereamino acid 15 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, or Asp. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20is Ala, Gly, Val, Leu, or Ile; and where amino acid 15 is Ala, Gly, Val,Leu, or ile. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg,or His; and where amino acid 15 is Ala, Gly, Val, Leu, or Ile. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2J, where amino acid 42 is Ala, amino acid 20 is Ala,and amino acid 15 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2J, where aminoacid 42 is Ala, amino acid 20 is Gly, and amino acid 15 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2J, where amino acid 42 is Val, amino acid 20 is Ala,and amino acid 15 is Gly. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 21, where aminoacid 42 is Leu, amino acid 20 is Ala, and amino acid 15 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2J, where amino acid 42 is Ile, amino acid 20 is Ala,and amino acid 15 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2J, where aminoacid 42 is Ala, amino acid 20 is Asn, and amino acid 15 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 21, where amino acid 42 is Ala, amino acid 20 is Gln,and amino acid 15 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2J, where aminoacid 42 is Ala, amino acid 20 is Lys, and amino acid 15 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2I, where amino acid 42 is Ala, amino acid 20 is Arg,and amino acid 15 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 21, where aminoacid 42 is Ala, amino acid 20 is His, and amino acid 15 is Ala. In somecases, the F42/D20/E15 substitution variant IL-2 polypeptides describedabove have a binding affinity for IL2R that is from about 100 nM to 150nM, from about 150 nM to about 200 nM, from about 200 nM to about 250nM, from about 250 nM to about 300 nM, from about 300 nM to about 350nM, from about 350 nM to about 400 nM, from about 400 nM to about 500nM, from about 500 nM to about 600 nM, from about 600 nM to about 700nM, from about 700 nM to about 800 nM, from about 800 nM to about 900nM, from about 900 nM to about 1 μM, to about 1 μm to about 5 μM, fromabout 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μMto about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM toabout 100 μM. In some cases, such variant IL-2 polypeptides bind IL2Rwith a binding affinity that is at least 5%, 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50% less, at least 55% less, at least 60% less, at least65% less, at least 70% less, at least 75% less, at least 80% less, atleast 85% less, at least 90% less, at least 95% less, or more than 95%less, than the binding affinity of an IL-2 polypeptide comprising theamino acid sequence depicted in FIG. 2A for an IL2R (e.g., an IL2Rcomprising alpha, beta, and gamma chains comprising the amino acidsequences (mature forms) depicted in FIG. 3A-3C). In some cases, suchvariant IL-2 polypeptide has a length of 133 amino acids.

F42, D20, and H16 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2K, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu; where amino acid 20 is an amino acid other than an asparticacid, e.g., where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu; and whereamino acid 16 is an amino acid other than a histidine, e.g., where aminoacid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys,Met, Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2K,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2K, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg,or His; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2K, where aminoacid 42 is Ala, amino acid 20 is Gly, and amino acid 16 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Val, amino acid 20 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2K, where aminoacid 42 is Leu, amino acid 20 is Ala, and amino acid 16 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ile, amino acid 20 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2K, where aminoacid 42 is Ala, amino acid 20 is Asn, and amino acid 16 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Gln,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2K, where aminoacid 42 is Ala, amino acid 20 is Lys, and amino acid 16 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Arg,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2K, where aminoacid 42 is Ala, amino acid 20 is His, and amino acid 16 is Ala. In somecases, the F42/D20/H16 substitution variant IL-2 polypeptides describedabove have a binding affinity for IL2R that is from about 100 nM to 150nM, from about 150 nM to about 200 nM, from about 200 nM to about 250nM, from about 250 nM to about 300 nM, from about 300 nM to about 350nM, from about 350 nM to about 400 nM, from about 400 nM to about 500nM, from about 500 nM to about 600 nM, from about 600 nM to about 700nM, from about 700 nM to about 800 nM, from about 800 nM to about 900nM, firm about 900 nM to about 1 μM, to about 1 μM to about 5 μM, fromabout 5 μM to about 10 μM, from about 10 M to about 15 μM, from about 15μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM toabout 50 μM, from about 50 μM to about 75 μM, or from about 75 μM toabout 100 μM. In some cases, such variant IL-2 polypeptides bind IL2Rwith a binding affinity that is at least 5%, 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50% less, at least 55% less, at least 60% less, at least65% less, at least 70% less, at least 75% less, at least 80% less, atleast 85% less, at least 90% less, at least 95% less, or more than 95%less, than the binding affinity of an IL-2 polypeptide comprising theamino acid sequence depicted in FIG. 2A for an IL2R (e.g., an IL2Rcomprising alpha, beta, and gamma chains comprising the amino acidsequences (mature forms) depicted in FIG. 3A-3C). In some cases, suchvariant IL-2 polypeptide has a length of 133 amino acids.

F42, D20, and Q126 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2L, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His.Asp, or Glu; where amino acid 20 is an amino acid other than an asparticacid, e.g., where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met. Asn, Gln, Lys, Arg, His, or Glu; and whereamino acid 126 is an amino acid other than a glutamine, e.g., whereamino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2L,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20is Ala, Gly, Val, Leu, or Ile; and where amino acid 126 is Ala, Gly,Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln,Lys, Arg, or His; and where amino acid 126 is Ala, Gly, Val, Leu, orIle. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2L, where amino acid 42 is Ala, aminoacid 20 is Ala, and amino acid 126 is Ala. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2L,where amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 126 isGly. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2L, where amino acid 42 is Val, aminoacid 20 is Ala, and amino acid 126 is Gly. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2L,where amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 126 isGly. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2L, where amino acid 42 is ile, aminoacid 20 is Ala, and amino acid 126 is Ala. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2L,where amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 126 isAla. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2L, where amino acid 42 is Ala, aminoacid 20 is Gln, and amino acid 126 is Ala. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2L,where amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 126 isAla. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2L, where amino acid 42 is Ala, aminoacid 20 is Arg, and amino acid 126 is Ala. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2L,where amino acid 42 is Ala, amino acid 20 is His, and amino acid 126 isAla. In some cases, the F42/D20/Q126 substitution variant IL-2polypeptides described above have a binding affinity for IL2R that isfrom about 100 nM to 150 nM, from about 150 nM to about 200 nM, fromabout 200 nM to about 250 nM, from about 250 nM to about 300 nM, fromabout 300 nM to about 350 nM, from about 350 nM to about 400 nM, fromabout 400 nM to about 500 nM, from about 500 nM to about 600 nM, fromabout 600 nM to about 700 nM, from about 700 nM to about 800 nM, fromabout 800 nM to about 900 nM, from about 900 nM to about 1 μM, to about1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM toabout 15 μM, from about 15 iμM to about 20 μM, from about 20 μM to about25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM,or from about 75 μM to about 100 μM. In some cases, such variant IL-2polypeptides bind IL2R with a binding affinity that is at least 5%, 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50% less, at least 55% less, at least60% less, at least 65% less, at least 70% less, at least 75% less, atleast 80% less, at least 85% less, at least 90% less, at least 95% less,or more than 95% less, than the binding affinity of an IL-2 polypeptidecomprising the amino acid sequence depicted in FIG. 2A for an IL2R(e.g., an IL2R comprising alpha, beta, and gamma chains comprising theamino acid sequences (mature forms) depicted in FIG. 3A-3C). In somecases, such variant IL-2 polypeptide has a length of 133 amino acids.

F42, D20, and Y45 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2M, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His.Asp, or Glu; where amino acid 20 is an amino acid other than an asparticacid, e.g., where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met. Asn, Gln, Lys, Arg, His, or Glu; and whereamino acid 45 is an amino acid other than a tyrosine, e.g., where aminoacid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His. Asp, or Glu. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2M,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20is Ala, Gly, Val, Leu, or Ile; and where amino acid 45 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2M, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg,or His; and where amino acid 45 is Ala, Gly, Val, Leu, or Ile. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Ala,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2M, where aminoacid 42 is Ala, amino acid 20 is Gly, and amino acid 45 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Val, amino acid 20 is Ala,and amino acid 45 is Gly. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2M, where aminoacid 42 is Leu, amino acid 20 is Ala, and amino acid 45 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ile, amino acid 20 is Ala,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2M, where aminoacid 42 is Ala, amino acid 20 is Asn, and amino acid 45 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Gln,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2M, where aminoacid 42 is Ala, amino acid 20 is Lys, and amino acid 45 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Arg,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2M, where aminoacid 42 is Ala, amino acid 20 is His, and amino acid 45 is Ala. In somecases, the F42/D20/Y45 substitution variant IL-2 polypeptides describedabove have a binding affinity for IL2R that is from about 100 nM to 150nM, from about 150 nM to about 200 nM, from about 200 nM to about 250nM, from about 250 nM to about 300 nM, from about 300 nM to about 350nM, from about 350 nM to about 400 nM, from about 400 nM to about 500nM, from about 500 nM to about 600 nM, from about 600 nM to about 700nM, from about 700 nM to about 800 nM, from about 800 nM to about 900nM, from about 900 nM to about 1 μM, to about 1 M to about 5 μM, fromabout 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μMto about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM toabout 100 μM. In some cases, such variant IL-2 polypeptides bind IL2Rwith a binding affinity that is at least 5%, 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50% less, at least 55% less, at least 60% less, at least65% less, at least 70% less, at least 75% less, at least 80% less, atleast 85% less, at least 90% less, at least 95% less, or more than 95%less, than the binding affinity of an IL-2 polypeptide comprising theamino acid sequence depicted in FIG. 2A for an IL2R (e.g., an IL2Rcomprising alpha, beta, and gamma chains comprising the amino acidsequences (mature forms) depicted in FIG. 3A-3C). In some cases, suchvariant IL-2 polypeptide has a length of 133 amino acids.

F42, D20, Y45, and H16 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2N, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu; where amino acid 20 is an amino acid other than an asparticacid, e.g., where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu; whereamino acid 45 is an amino acid other than a tyrosine, e.g., where aminoacid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His, Asp, or Glu; and where amino acid 16 is anamino acid other than a histidine, e.g., where amino acid 16 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met. Asn, Gln,Lys, Arg. Asp, or Glu. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2N, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly,Val, Leu, or Ile; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; andwhere amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2N, where amino acid 42 is Ala, Gly, Val, Leu, or Ile;where amino acid 20 is Asn, Gln, Lys, Arg, or His; where amino acid 45is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 16 isAla. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2N, where amino acid 42 is Ala, aminoacid 20 is Gly, amino acid 45 is Gly, and amino acid 16 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2N, where amino acid 42 is Val, amino acid 20 is Ala,amino acid 45 is Gly, and amino acid 16 is Ala. In some cases, a variantIL-2 polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2N, where amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45is Gly, and amino acid 16 is Val. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2N,where amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2N, where aminoacid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and aminoacid 16 is Ala. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, amino acid 20 is Gln, amino acid 45 is Ala, and amino acid 16 isAla. In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2N, where amino acid 42 is Ala, aminoacid 20 is Lys, amino acid 45 is Ala, and amino acid 16 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2N, where amino acid 42 is Ala, amino acid 20 is Arg,amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, a variantIL-2 polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2N, where amino acid 42 is Ala, amino acid 20 is His, amino acid 45is Ala, and amino acid 16 is Ala. In some cases, the F42/D20/Y45/H16substitution variant IL-2 polypeptides described above have a bindingaffinity for IL2R that is from about 100 nM to 150 nM, from about 150 nMto about 200 nM, from about 200 nM to about 250 μM, from about 250 nM toabout 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 700 nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM,from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 μM. In somecases, such variant IL-2 polypeptides bind IL2R with a binding affinitythat is at least 5%, 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% less,at least 55% less, at least 60% less, at least 65% less, at least 70%less, at least 75% less, at least 80% less, at least 85% less, at least90% less, at least 95% less, or more than 95% less, than the bindingaffinity of an IL-2 polypeptide comprising the amino acid sequencedepicted in FIG. 2A for an IL2R (e.g., an IL2R comprising alpha, beta,and gamma chains comprising the amino acid sequences (mature forms)depicted in FIG. 3A-3C). In some cases, such variant IL-2 polypeptidehas a length of 133 amino acids.

F42, D20, Y45, and Q126 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2O, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His.Asp, or Glu; where amino acid 20 is an amino acid other than an asparticacid, e.g., where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu; whereamino acid 45 is an amino acid other than a tyrosine, e.g., where aminoacid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met.Asn, Gln, Lys, Arg, His, Asp, or Glu; and where amino acid 126 is anamino acid other than a glutamine, e.g., where amino acid 126 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys,Arg, His. Asp, or Glu. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly,Val, Leu, or Ile; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; andwhere amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2O, where amino acid 42 is Ala, Gly, Val, Leu, or Ile;where amino acid 20 is Asn, Gln, Lys, Arg, or His; where amino acid 45is Ala, Gly, Val, Leu, or Ile; and where amino acid 126 is Ala, Gly,Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, and aminoacid 126 is Val. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, and aminoacid 126 is Gly. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2O, where aminoacid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, the F42/D20/Y45/Q126 substitutionvariant IL-2 polypeptides described above have a binding affinity forIL2R that is from about 100 nM to 150 nM, from about 150 nM to about 200nM, from about 200 nM to about 250 nM, from about 250 nM to about 300nM, from about 300 nM to about 350 nM, from about 350 nM to about 400nM, from about 400 nM to about 500 nM, from about 500 nM to about 600nM, from about 600 nM to about 700 nM, from about 700 nM to about 800nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM,to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μMto about 25 μM, from about 25 μM to about 50 μM, from about 50 μM toabout 75 μM, or from about 75 μM to about 100 μM. In some cases, suchvariant IL-2 polypeptides bind IL2R with a binding affinity that is atleast 5%, 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50% less, at least55% less, at least 60% less, at least 65% less, at least 70% less, atleast 75% less, at least 80% less, at least 85% less, at least 90% less,at least 95% less, or more than 95% less, than the binding affinity ofan IL-2 polypeptide comprising the amino acid sequence depicted in FIG.2A for an IL2R (e.g., an IL2R comprising alpha, beta, and gamma chainscomprising the amino acid sequences (mature forms) depicted in FIG.3A-3C). In some cases, such variant IL-2 polypeptide has a length of 133amino acids.

F42, D20, Y45, H16, and Q126 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2P, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp. Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His,Asp, or Glu; where amino acid 20 is an amino acid other than an asparticacid, e.g., where amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe,Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, or Glu; whereamino acid 45 is an amino acid other than a tyrosine, e.g., where aminoacid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met,Asn, Gln, Lys, Arg, His. Asp, or (Glu; where amino acid 126 is an aminoacid other than a glutamine, e.g., where amino acid 126 is Gly, Ala,Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg,His, Asp, or Glu; and where amino acid 16 is an amino acid other than ahistidine, e.g., where amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. Insome cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2P, where amino acid 42 is Ala, Gly, Val,Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu, or Ile; whereamino acid 45 is Ala, Gly, Val, Leu, or Ile; where amino acid 126 isAla, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2P, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg,or His; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; where aminoacid 126 is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2P, where aminoacid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, amino acid126 is Ala, and amino acid 16 is Ala. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly,amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2P, where amino acid 42 is Val, amino acid 20 is Ala,amino acid 45 is Gly, amino acid 126 is Ala, and amino acid 16 is Ala.In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2P, where amino acid 42 is Leu, aminoacid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Val, and aminoacid 16 is Ala. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2P, where amino acid 42 isile, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Gly,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2P, where aminoacid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, amino acid126 is Ala, and amino acid 16 is Ala. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala,amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2P, where amino acid 42 is Ala, amino acid 20 is Lys,amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala.In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2P, where amino acid 42 is Ala, aminoacid 20 is Arg, amino acid 45 is Ala, amino acid 126 is Ala, and aminoacid 16 is Ala. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2P, where amino acid 42 isAla, amino acid 20 is His, amino acid 45 is Ala, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, the F42/D20/Y45/H16/Q126substitution variant IL-2 polypeptides described above have a bindingaffinity for IL2R that is from about 100 nM to 150 nM, from about 150 nMto about 200 nM, from about 200 nM to about 250 nM, from about 250 nM toabout 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 700 nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM,from about 10 μM to about 15 μM, from about 15 μM to about 20 tμM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 μM. In somecases, such variant IL-2 polypeptides bind IL2R with a binding affinitythat is at least 5%, 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50% less,at least 55% less, at least 60% less, at least 65% less, at least 70%less, at least 75% less, at least 80% less, at least 85% less, at least90% less, at least 95% less, or more than 95% less, than the bindingaffinity of an IL-2 polypeptide comprising the amino acid sequencedepicted in FIG. 2A for an IL2R (e.g., an IL2R comprising alpha, beta,and gamma chains comprising the amino acid sequences (mature forms)depicted in FIG. 3A-3C). In some cases, such variant IL-2 polypeptidehas a length of 133 amino acids.

F42, Q126, and H16 Substitutions

In some cases, a variant IL-2 polypeptide of the present disclosurecomprises an amino acid sequence having at least 90%, at least 95%, atleast 98%, or at least 99%, amino acid sequence identity to the aminoacid sequence depicted in FIG. 2Q, where amino acid 42 is an amino acidother than a phenylalanine, e.g., where amino acid 42 is Gly, Ala, Val,Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His.Asp, or Glu; where amino acid 126 is an amino acid other than aglutamine, e.g., where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro,Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu; andwhere amino acid 16 is an amino acid other than a histidine, e.g., whereamino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, a variant IL-2polypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2Q,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 126is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2Q, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 126 is Asn, Gln, Lys, Arg,or His; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2Q, where aminoacid 42 is Ala, amino acid 126 is Gly, and amino acid 16 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Val, amino acid 126 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2Q, where aminoacid 42 is Leu, amino acid 126 is Ala, and amino acid 16 is Gly. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ile, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2Q, where aminoacid 42 is Ala, amino acid 126 is Asn, and amino acid 16 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2Q, where aminoacid 42 is Ala, amino acid 126 is Lys, and amino acid 16 is Ala. In somecases, a variant IL-2 polypeptide of the present disclosure comprises anamino acid sequence having at least 90%, at least 95%, at least 98%, orat least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Arg,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2Q, where aminoacid 42 is Ala, amino acid 126 is His, and amino acid 16 is Ala. In somecases, the F42/Q126/H16 substitution variant IL-2 polypeptides have abinding affinity for IL2R that is from about 100 nM to 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 500 nM, fromabout 500 nM to about 600 nM, from about 600 nM to about 700 nM, fromabout 700 nM to about 800 nM, from about 800 nM to about 900 nM, fromabout 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μMto about 10 μM, from about 10 μM to about 15 μM, from about 15 μM toabout 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100μM. In some cases, such variant IL-2 polypeptides bind IL2R with abinding affinity that is at least 5%, 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50% less, at least 55% less, at least 60% less, at least 65% less,at least 70% less, at least 75% less, at least 80% less, at least 85%less, at least 90% less, at least 95% less, or more than 95% less, thanthe binding affinity of an IL-2 polypeptide comprising the amino acidsequence depicted in FIG. 2A for an IL2R (e.g., an IL2R comprisingalpha, beta, and gamma chains comprising the amino acid sequences(mature forms) depicted in FIG. 3A-3C). In some cases, such variant IL-2polypeptide has a length of 133 amino acids.

Fusion Polypeptides

The present disclosure provides IL-2 fusion polypeptides. A fusionpolypeptide of the present disclosure comprises: a) a variant IL-2polypeptide of the present disclosure; and b) a heterologous fusionpartner. In some cases, the heterologous fusion partner is fused to theN-terminus of the variant IL-2 polypeptide. In some cases, theheterologous fusion partner is fused to the C-terminus of the variantIL-2 polypeptide. In some cases, an IL-2 fusion polypeptide of thepresent disclosure comprises a first heterologous fusion partner fusedto the N-terminus of the variant IL-2 polypeptide, and a secondheterologous fusion partner fused to the C-terminus of the variant IL-2polypeptide.

The total length of an IL-2 fusion polypeptide of the present disclosurecan range from 135 amino acids to 2000 amino acids. For example, an IL-2fusion polypeptide of the present disclosure can range from 135 aminoacids to 150 amino acids, from 150 amino acids to 175 amino acids, from175 amino acids to 200 amino acids, from 200 amino acids to 225 aminoacids, from 225 amino acids to 250 amino acids, from 250 amino acids to275 amino acids, from 275 amino acids to 300 amino acids, from 300 aminoacids to 350 amino acids, from 350 amino acids, from 350 amino acids to400 amino acids, from 400 amino acids, from 400 amino acids to 450 aminoacids, from 450 amino acids to 500 amino acids, from 500 amino acids to600 amino acids, from 600 amino acids to 700 amino acids, from 700 aminoacids to 800 amino acids, from 800 amino acids to 900 amino acids, from900 amino acids to 1000 amino acids, from 1000 amino acids to 1250 aminoacids, from 1250 amino acids to 1500 amino acids, from 1500 amino acidsto 1750 amino acids, or from 1750 amino acids to 2000 amino acids.

Suitable fusion partners include, hut are not limited to, atransmembrane domain; an antibody Fc region; an antigen-binding regionof an antibody; a cytokine (other than IL-2); an immunomodulatorydomain; an intracellular signaling domain; and the like.

T-Cell Modulatory Multimeric Polypeptides

The present disclosure provides multimeric (e.g., heterodimeric,heterotrimeric) polypeptides. The multimeric polypeptides are T cellmodulatory polypeptides, and are also referred to herein as “T-cellmodulatory multimeric polypeptides,” or “synTac” (for “immunologicalsynapse for T cell activation”). FIG. 1A-1D provide schematic depictionsof T-cell modulatory multimeric polypeptides of the present disclosure.A T-cell modulatory multimeric polypeptide of the present disclosure isalso referred to as an “IL-2/synTac,” a “synTac polypeptide” or a“multimeric polypeptide.”

In some cases, a synTac polypeptide of the present disclosure comprisesa wild-type IL-2 polypeptide. In some cases, a synTac polypeptide of thepresent disclosure comprises a single copy of a wild-type IL-2polypeptide. In some cases, a synTac polypeptide of the presentdisclosure comprises two copies of a wild-type IL-2 polypeptide. In somecases, a synTac polypeptide of the present disclosure comprises threecopies of a wild-type IL-2 polypeptide. In some cases, the wild-typeIL-2 polypeptide comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2A.

In some cases, a synTac polypeptide of the present disclosure comprisesa variant IL-2 polypeptide of the present disclosure. As noted above, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure exhibits reduced binding affinity to an IL-2R,compared to the binding affinity of wild-type IL-2 to the IL-2R. Amultimeric polypeptide of the present disclosure that comprises avariant IL-2 polypeptide of the present disclosure also exhibits reducedbinding affinity for an IL-2R, compared to a control multimericpolypeptide comprising a wild-type IL-2 for IL-2R (e.g., an IL-2Rcomprising alpha, beta, and gamma polypeptides comprising the amino acidsequences (mature form) depicted in FIG. 3A-3C).

In some cases, a synTac polypeptide of the present disclosure exhibitsreduced binding affinity to IL-2R, compared to the binding affinity ofan IL-2 polypeptide comprising the amino acid sequence depicted in FIG.2A for IL-2R. For example, in some cases, a synTac polypeptide of thepresent disclosure binds IL-2R with a binding affinity that is less thanthe binding affinity of a control synTac polypeptide comprising an IL-2polypeptide comprising the amino acid sequence depicted in FIG. 2A foran IL-2R comprising alpha, beta and gamma polypeptides comprising theamino acid sequences (mature form) depicted in FIG. 3A-3C. For example,in some cases, a synTac polypeptide of the present disclosure bindsIL-2R with a binding affinity that is at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50% less, at least 55% less, at least 60% less, atleast 65% less, at least 70% less, at least 75% less, at least 80% less,at least 85% less, at least 90% less, at least 95% less, or more than95% less, than the binding affinity of a control synTac polypeptidecomprising an IL-2 polypeptide comprising the amino acid sequencedepicted in FIG. 2A for IL-2R (e.g., an IL-2R comprising alpha, beta,and gamma polypeptides comprising the amino acid sequences (mature form)depicted in FIG. 3A-3C).

In some cases, a synTac polypeptide of the present disclosure has abinding affinity for IL-2R that is from 100 nm to about 100 μM. In somecases, a synTac polypeptide of the present disclosure has a bindingaffinity for IL-2R that is from about 100 nM to 500 nM. For example, insome cases, a synTac polypeptide of the present disclosure has a bindingaffinity for IL-2R (e.g., an IL-2R comprising alpha, beta, and gammapolypeptides comprising the amino acid sequences (mature form) depictedin FIG. 3A-3C) that is from about 100 nM to about 150 nM, from about 150nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nMto about 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 450 nM, or from about 450 nM toabout 500 nM. In some cases, a synTac polypeptide of the presentdisclosure has a binding affinity for IL-2R (e.g., an IL-2R comprisingalpha, beta, and gamma polypeptides comprising the amino acid sequences(mature form) depicted in FIG. 3A-3C) that is from about 500 nM to 1 μM.For example, in some cases, a synTac polypeptide of the presentdisclosure has a binding affinity for IL-2R (e.g., an IL-2R comprisingalpha, beta, and gamma polypeptides comprising the amino acid sequences(mature form) depicted in FIG. 3A-3C) that is from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, or from about 900 nM to about1 μM. In some cases, a synTac polypeptide of the present disclosure hasa binding affinity for IL-2R (e.g., an IL-2R comprising alpha, beta, andgamma polypeptides comprising the amino acid sequences (mature form)depicted in FIG. 3A-3C) that is from about 1 μM to 10 μM. For example,in some cases, a synTac polypeptide of the present disclosure has abinding affinity for IL-2R (e.g., an IL-2R comprising alpha, beta, andgamma polypeptides comprising the amino acid sequences (mature form)depicted in FIG. 3A-3C) that is from about 1 μM to 2 μM, from about 2 μMto about 3 μM, from about 3 μM to about 4 μM, from about 4 μM to about 5μM, from about 5 μM to about 6 μM, from about 6 μM to about 7 μM, fromabout 7 μM to about 8 μM, from about 8 M to about 9 μM, or from about 9μM to about 10 μM. In some cases, a synTac polypeptide of the presentdisclosure has a binding affinity for IL-2R (e.g., an IL-2R comprisingalpha, beta, and gamma polypeptides comprising the amino acid sequences(mature form) depicted in FIG. 3A-3C) that is from about 10 μM to 100μM. For example, in some cases, a synTac polypeptide of the presentdisclosure has a binding affinity for IL-2R (e.g., an IL-2R comprisingalpha, beta, and gamma polypeptides comprising the amino acid sequences(mature form) depicted in FIG. 3A-3C) that is from about 10 μM to about20 μM, from about 20 μM to about 30 μM, from about 30 μM to about 40 μM,from about 40 M to about 50 μM, from about 50 μM to about 60 μM, fromabout 60 μM to about 70 μM, from about 70 μM to about 80 μM, from about80 μM to about 90 μM, or from about 90 μM to about 100 μM.

Determining Binding Affinity

Binding affinity between an immunomodulatory polypeptide and its cognateco-immunomodulatory polypeptide can be determined by bio-layerinterferometry (BLI) using purified immunomodulatory polypeptide andpurified cognate co-immunomodulatory polypeptide. Binding affinitybetween a synTac of the present disclosure and its cognateco-immunomodulatory polypeptide can also be determined by BLI usingpurified synTac and the cognate co-immunomodulatory polypeptide. BLImethods are well known to those skilled in the art. See, e.g., Lad etal. (2015) J. Biomol. Screen. 20(4):498-507; and Shah and Duncan (2014)J. Vis. Exp. 18:e51383. The specific and relative binding affinitiesdescribed in this disclosure between an immunomodulatory polypeptide andits cognate co-immunomodulatory polypeptide, or between a synTac and itscognate co-immunomodulatory polypeptide, can be determined using thefollowing procedures.

To determine binding affinity between a synTac of the present disclosureand its cognate co-immunomodulatory polypeptide, a BLI assay can becarried out using an Octet RED 96 (Pal FortéBio) instrument, or asimilar instrument, as follows. To determine binding affinity of aT-cell modulatory multimeric polypeptide (e.g., a synTac of the presentdisclosure; or a control T-cell modulatory multimeric polypeptide (wherea control T-cell modulatory multimeric polypeptide comprises a wild-typeimmunomodulatory polypeptide)), the T-cell modulatory multimericpolypeptide is immobilized onto an insoluble support (a “biosensor”).The immobilized T-cell modulatory multimeric polypeptide is the“target.” Immobilization can be effected by immobilizing a captureantibody onto the insoluble support, where the capture antibodyimmobilizes the T-cell modulatory multimeric polypeptide. For example,immobilization can be effected by immobilizing anti-Fe (e.g., anti-humanIgG Fc) antibodies onto the insoluble support, where the immobilizedanti-Fc antibodies bind to and immobilize the T-cell modulatorymultimeric polypeptide (where the T-cell modulatory multimericpolypeptide comprises an IgFc polypeptide). A co-immunomodulatorypolypeptide is applied, at several different concentrations, to theimmobilized T-cell modulatory multimeric polypeptide, and theinstrument's response recorded. Assays are conducted in a liquid mediumcomprising 25 mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl,0.1% bovine serum albumin, and 0.02% Tween 20 nonionic detergent.Binding of the co-immunomodulatory polypeptide to the immobilized T-cellmodulatory multimeric polypeptide is conducted at 30° C. As a positivecontrol for binding affinity, an anti-MHC Class I monoclonal antibodycan be used. For example, anti-HLA Class I monoclonal antibody W6/32(American Type Culture Collection No. HB-95; Parham et al. (1979) J.Immunol. 123:342), which has a K_(D) of 7 nM, can be used. A standardcurve can be generated using serial dilutions of the anti-MHC Class Imonoclonal antibody. The co-immunomodulatory polypeptide, or theanti-MHC Class I mAb, is the “analyte.” BLI analyzes the interferencepattern of white light reflected from two surfaces: i) from theimmobilized polypeptide (“target”); and ii) an internal reference layer.A change in the number of molecules (“analyte”; e.g.,co-immunomodulatory polypeptide; anti-HLA antibody) bound to thebiosensor tip causes a shift in the interference pattern; this shift ininterference pattern can be measured in real time. The two kinetic termsthat describe the affinity of the target/analyte interaction are theassociation constant (k_(a)) and dissociation constant (k_(d)). Theratio of these two terms (k_(d)/_(a)) gives rise to the affinityconstant K_(D).

As noted above, determining binding affinity between an immunomodulatorypolypeptide (e.g., IL-2 or an IL-2 variant) and its cognateco-immunomodulatory polypeptide (e.g., IL-2R) also can be determined byBLI. The assay is similar to that described above for the synTacmultimeric polypeptide. A BLI assay can be carried out using an OctetRED 96 (Pal FortéBio) instrument, or a similar instrument, as follows. Acomponent immunomodulatory polypeptide of a synTac of the presentdisclosure (e.g., a variant IL-2 polypeptide of the present disclosure);and a control immunomodulatory polypeptide (where a controlimmunomodulatory polypeptide comprises a wild-type immunomodulatorypolypeptide, e.g. wild-type IL-2)) are immobilized onto an insolublesupport (a “biosensor”). The immunomodulatory polypeptide is the“target.” Immobilization can be effected by immobilizing a captureantibody onto the insoluble support, where the capture antibodyimmobilizes the immunomodulatory polypeptide. For example, if the targetis fused to an immuno-affinity tag (e.g. FLAG, human IgG Fe)immobilization can be effected by immobilizing with the appropriateantibody to the immuno-affinity tag (e.g., anti-human IgG Fc) onto theinsoluble support, where the immobilized antibodies bind to andimmobilize the immunomodulatory polypeptide (where the immunomodulatorypolypeptide comprises an IgFc polypeptide). A co-immunomodulatorypolypeptide (or polypeptides) is applied, at several differentconcentrations, to the immobilized immunomodulatory polypeptide, and theinstrument's response recorded. Alternatively, a co-immunomodulatorypolypeptide (or polypeptides) is immobilized to the biosensor (e.g., forthe IL-2 receptor heterotrimer, as a monomeric subunit, heterodimericsubcomplex, or the complete heterotrimer) and the immunomodulatorypolypeptide is applied, at several different concentrations, to theimmobilized coimmunomodulatory polypeptide(s), and the instrument'sresponse is recorded. Assays are conducted in a liquid medium comprising25 mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1%bovine serum albumin, and 0.02% Tween 20 nonionic detergent. Binding ofthe co-immunomodulatory polypeptide to the immobilized immunomodulatorypolypeptide is conducted at 30° C. As a positive control for bindingaffinity, an anti-MHC Class I monoclonal antibody can be used. Forexample, anti-HLA Class I monoclonal antibody W6/32 (American TypeCulture Collection No. HB-95; Parham et al. (1979) J. Immunol. 123:342),which has a K_(D) of 7 nM, can be used. A standard curve can begenerated using serial dilutions of the anti-MHC Class I monoclonalantibody. The co-immunomodulatory polypeptide, or the anti-MHC Class ImAb, is the “analyte.” BLI analyzes the interference pattern of whitelight reflected from two surfaces: i) from the immobilized polypeptide(“target”); and ii) an internal reference layer. A change in the numberof molecules (“analyte”; e.g., co-immunomodulatory polypeptide; anti-HLAantibody) bound to the biosensor tip causes a shift in the interferencepattern; this shift in interference pattern can be measured in realtime. The two kinetic terms that describe the affinity of thetarget/analyte interaction are the association constant (k_(a)) anddissociation constant (k_(d)). The ratio of these two terms (k_(d)/_(a))gives rise to the affinity constant K_(D). Determining the bindingaffinity of both a wild-type immunomodulatory polypeptide (e.g., IL-2)for its receptor (e.g., IL-2R) and a variant immunomodulatorypolypeptide (e.g., an IL-2 variant as disclosed herein) for its cognateco-immunomodulatory polypeptide (e.g., its receptor) (e.g., IL-2R) thusallows one to determine the relative binding affinity of the variantco-immunomodulatory polypeptide, as compared to the wild-typeco-immunomodulatory polypeptide, for the cognate co-immunomodulatorypolypeptide. That is, one can determine whether the binding affinity ofa variant immunomodulatory polypeptide for its receptor (its cognateco-immunomodulatory polypeptide) is reduced as compared to the bindingaffinity of the wild-type immunomodulatory polypeptide for the samecognate co-immunomodulatory polypeptide, and, if so, what is thepercentage reduction from the binding affinity of the wild-typeco-immunomodulatory polypeptide.

The BLI assay is carried out in a multi-well plate. To run the assay,the plate layout is defined, the assay steps are defined, and biosensorsare assigned in Octet Data Acquisition software. The biosensor assemblyis hydrated. The hydrated biosensor assembly and the assay plate areequilibrated for 10 minutes on the Octet instrument. Once the data areacquired, the acquired data are loaded into the Octet Data Analysissoftware. The data are processed in the Processing window by specifyingmethod for reference subtraction, y-axis alignment, inter-stepcorrection, and Savitzky-Golay filtering. Data are analyzed in theAnalysis window by specifying steps to analyze (Association andDissociation), selecting curve fit model (1:1), fitting method (global),and window of interest (in seconds). The quality of fit is evaluated.K_(D) values for each data trace (analyte concentration) can be averagedif within a 3-fold range. K_(D) error values should be within one orderof magnitude of the affinity constant values; R² values should be above0.95. See, e.g., Abdiche et al. (2008) J. Anal. Biochem. 377:209.

In some cases, the ratio of: i) the binding affinity of a control T-cellmodulatory multimeric polypeptide (where the control comprises awild-type immunomodulatory polypeptide, e.g., wild-type IL-2) to acognate co-immunomodulatory polypeptide (e.g., IL-2R) to ii) the bindingaffinity of a T-cell modulatory multimeric polypeptide of the presentdisclosure comprising a variant of the wild-type immunomodulatorypolypeptide (e.g., variant IL-2) to the cognate co-immunomodulatorypolypeptide (e.g., IL-2R), when measured by BLI (as described above), isat least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, atleast 500:1, at least 10²:1, at least 5×10²:1, at least 10³:1, at least5×10⁴:1, at least 10⁴:1, at least 10⁵:1, or at least 10⁶:1. In somecases, the ratio of: i) the binding affinity of a control T-cellmodulatory multimeric polypeptide (where the control comprises awild-type immunomodulatory polypeptide) to a cognate co-immunomodulatorypolypeptide to ii) the binding affinity of a T-cell modulatorymultimeric polypeptide of the present disclosure comprising a variant ofthe wild-type immunomodulatory polypeptide to the cognateco-immunomodulatory polypeptide, when measured by BLI, is in a range offrom 1.5:1 to 10⁶:1, e.g., from 1.5:1 to 10:1, from 10:1 to 50:1, from50:1 to 10²:1, from 10²:1 to 10³:1, from 10³:1 to 10⁴:1, from 10⁴:1 to10⁵:1, or from 10⁵:1 to 10⁶:1.

In some cases, the ratio of: i) the binding affinity of a controlimmunomodulatory polypeptide (where the control comprises a wild-typeimmunomodulatory polypeptide, e.g., wild-type IL-2) to a cognateco-immunomodulatory polypeptide (e.g., TL-2R) to ii) the bindingaffinity of a immunomodulatory polypeptide of the present disclosurecomprising a variant of the wild-type immunomodulatory polypeptide(e.g., variant IL-2) to the cognate co-immunomodulatory polypeptide(e.g., IL-2R), when measured by BLI (as described above), is at least1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, atleast 20:1, at least 25:1, at least 50:1, at least 100:1, at least500:1, at least 10²:1, at least 5×10²:1, at least 10³:1, at least5×10³:1, at least 10⁴:1, at least 10⁵:1, or at least 10⁶:1. In somecases, the ratio of: i) the binding affinity of a controlimmunomodulatory polypeptide (where the control comprises a wild-typeimmunomodulatory polypeptide) to a cognate co-immunomodulatorypolypeptide to ii) the binding affinity of a immunomodulatorypolypeptide of the present disclosure comprising a variant of thewild-type immunomodulatory polypeptide to the cognateco-immunomodulatory polypeptide, when measured by BLI, is in a range offrom 1.5:1 to 10⁶:1, e.g., from 1.5:1 to 10:1, from 10:1 to 50:1, from50:1 to 10²:1, from 10²:1 to 10³:1, from 10³:1 to 10⁴:1, from 10⁴:1 to10⁵:1, or from 10⁵:1 to 10⁶:1.

A variant IL-2 polypeptide present in a synTac polypeptide of thepresent disclosure can have a single amino acid substitution relative toa wild-type IL-2 polypeptide (e.g., a IL-2 polypeptide comprising theamino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO:1).In some cases, a variant IL-2 polypeptide present in a synTacpolypeptide of the present disclosure has from 2 to 10 amino acidsubstitutions relative to a wild-type IL-2 polypeptide (e.g., a IL-2polypeptide comprising the amino acid sequence depicted in FIG. 2A or asset forth in SEQ ID NO:1). In some cases, a variant IL-2 polypeptidepresent in a synTac polypeptide of the present disclosure has 2 aminoacid substitutions relative to a wild-type IL-2 polypeptide (e.g., aIL-2 polypeptide comprising the amino acid sequence depicted in FIG. 2Aor as set forth in SEQ ID NO:1). In some cases, a variant IL-2polypeptide present in a synTac polypeptide of the present disclosurehas 3 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., a IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variant IL-2polypeptide present in a synTac polypeptide of the present disclosurehas 4 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., a IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variant IL-2polypeptide present in a synTac polypeptide of the present disclosurehas 5 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., a IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variant IL-2polypeptide present in a synTac polypeptide of the present disclosurehas 6 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., a IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variant IL-2polypeptide present in a synTac polypeptide of the present disclosurehas 7 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., a IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variant IL-2polypeptide present in a synTac polypeptide of the present disclosurehas 8 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., a IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variant IL-2polypeptide present in a synTac polypeptide of the present disclosurehas 9 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., a IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 2A or as set forth in SEQ ID NO:1). In some cases, a variant IL-2polypeptide present in a synTac polypeptide of the present disclosurehas 10 amino acid substitutions relative to a wild-type IL-2 polypeptide(e.g., a IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 2A or as set forth in SEQ ID NO:1).

In some cases, a multimeric polypeptide of the present disclosurecomprises a first polypeptide and a second polypeptide, where the firstpolypeptide comprises, in order from amino terminus (N-terminus) tocarboxyl terminus (C-terminus); a) an epitope (e.g., a T-cell epitope);b) a first major histocompatibility complex (MHC) polypeptide and c) animmunomodulatory polypeptide (e.g., a variant IL-2 polypeptide of thepresent disclosure); and where the second polypeptide comprises, inorder from N-terminus to C-terminus; a) a second MHC polypeptide; and b)an immunoglobulin (Ig) Fc polypeptide. In other cases, a multimericpolypeptide of the present disclosure comprises a first polypeptide anda second polypeptide, where the first polypeptide comprises, in orderfrom N-terminus to C-terminus; a) an epitope (e.g., a T-cell epitope);and b) a first MHC polypeptide; and where the second polypeptidecomprises, in order from N-terminus to C-terminus; a) animmunomodulatory polypeptide (e.g., a variant IL-2 polypeptide of thepresent disclosure); b) a second MHC polypeptide; and c) an Ig Fcpolypeptide. In some instances, the first and the second MHCpolypeptides are Class I MHC polypeptides; e.g., in some cases, thefirst MHC polypeptide is an MHC Class I β2-microglobulin (B2M or β2M)polypeptide, and the second MHC polypeptide is an MHC Class I heavychain (H chain); or the first MHC polypeptide is an MHC Class I H chain,and the second MHC polypeptide is an MHC Class I β2M polypeptide). Inother cases, the first and the second MHC polypeptides are Class II MHCpolypeptides; e.g., in some cases, the first MHC polypeptide is an MHCClass II α-chain polypeptide, and the second MHC polypeptide is an MHCClass II β-chain polypeptide. In other cases, the first polypeptide isan MHC Class II β-chain polypeptide, and the second MHC polypeptide isan MHC Class II α-chain polypeptide. In some cases, the multimericpolypeptide includes two or more immunomodulatory polypeptides, where atleast one of the immunomodulatory polypeptides is a variant IL-2immunomodulatory polypeptide of the present disclosure. Where amultimeric polypeptide of the present disclosure includes two or moreimmunomodulatory polypeptides, in some cases, the two or moreimmunomodulatory polypeptides are present in the same polypeptide chain,and may be in tandem. Where a multimeric polypeptide of the presentdisclosure includes two or more immunomodulatory polypeptides, in somecases, the two or more immunomodulatory polypeptides are present inseparate polypeptides. In some cases, a multimeric polypeptide of thepresent disclosure is a heterodimer. In some cases, a multimericpolypeptide of the present disclosure is a trimeric polypeptide.

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus; i) an epitope; and ii) a first MHC polypeptide; and b) asecond polypeptide comprising, in order from N-terminus to C-terminus;i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) animmunomodulatory domain (e.g., a variant IL-2 polypeptide of the presentdisclosure). In some cases, a multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus; i) an epitope; and ii) a first MHCpolypeptide; and b) a second polypeptide comprising, in order fromN-terminus to C-terminus; i) a second MHC polypeptide; and ii) animmunomodulatory domain (e.g., a variant IL-2 polypeptide of the presentdisclosure). In some cases, a multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus; i) an epitope; and ii) a first MHCpolypeptide; and b) a second polypeptide comprising, in order fromN-terminus to C-terminus; i) an immunomodulatory domain (e.g., a variantIL-2 polypeptide of the present disclosure); and ii) a second MHCpolypeptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus; i) an epitope; ii) a first MHC polypeptide;and iii) an immunomodulatory domain (e.g., a variant IL-2 polypeptide ofthe present disclosure); and b) a second polypeptide comprising, inorder from N-terminus to C-terminus; i) a second MHC polypeptide. Insome cases, where a multimeric polypeptide of the present disclosurecomprises a non-Ig scaffold, the non-Ig scaffold is an XTEN peptide, atransferrin polypeptide, an Fc receptor polypeptide, an elastin-likepolypeptide, a silk-like polypeptide, or a silk-elastin-likepolypeptide.

In some cases, a multimeric polypeptide of the present disclosure ismonovalent. In some cases, a multimeric polypeptide of the presentdisclosure is multivalent. In some cases, a multivalent multimericpolypeptide of the present disclosure comprises an immunoglobulin Fcpolypeptide on one of the first or the second polypeptide. For example,depending on the Fc polypeptide present in a multimeric polypeptide ofthe present disclosure, the multimeric polypeptide can be a homodimer,where two molecules of the multimeric polypeptide are present in thehomodimer, where the two molecules of the multimeric polypeptide can bedisulfide linked to one another, e.g., via the Fc polypeptide present inthe two molecules. As another example, a multimeric polypeptide of thepresent disclosure can comprise three, four, or five molecules of themultimeric polypeptide, where the molecules of the multimericpolypeptide can be disulfide linked to one another, e.g., via the Fcpolypeptide present in the molecules.

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus; i) an epitope; ii) a β2M polypeptide; and iii) a variantIL-2 polypeptide of the present disclosure; and b) a second polypeptidecomprising, in order from N-terminus to C-terminus; i) a Class I MHCheavy chain; and ii) an Fc polypeptide. In some cases, a multimericpolypeptide of the present disclosure comprises: a) a first polypeptidecomprising, in order from N-terminus to C-terminus; i) an epitope; andii) a β2M polypeptide; and b) a second polypeptide comprising, in orderfrom N-terminus to C-terminus; i) a variant IL-2 polypeptide of thepresent disclosure; ii) a Class I MHC heavy chain; and iii) an Fcpolypeptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus; i) an epitope; ii) a β2M polypeptide; iii) afirst variant IL-2 polypeptide of the present disclosure; iv) a secondvariant IL-2 polypeptide of the present disclosure; and v) a thirdvariant IL-2 polypeptide of the present disclosure; and b) a secondpolypeptide comprising, in order from N-terminus to C-terminus; i) aClass I MHC heavy chain; and ii) an Fc polypeptide. In some cases, thefirst, second, and third variant IL-2 polypeptides have the same aminoacid sequence. In some cases, the first, second, and third variant IL-2polypeptides differ from one another in amino acid sequence. In somecases, a multimeric polypeptide of the present disclosure comprises: a)a first polypeptide comprising, in order from N-terminus to C-terminus;i) an epitope; and ii) a β2M polypeptide; and b) a second polypeptidecomprising, in order from N-terminus to C-terminus; i) a first variantIL-2 polypeptide of the present disclosure; ii) a second variant IL-2polypeptide of the present disclosure; and iii) a third variant IL-2polypeptide of the present disclosure; iv) a Class I MHC heavy chain;and v) an Fc polypeptide. In some cases, the first, second, and thirdvariant TL-2 polypeptides have the same amino acid sequence. In somecases, the first, second, and third variant IL-2 polypeptides differfrom one another in amino acid sequence.

Linkers

A multimeric polypeptide of the present disclosure can include linkerpeptides interposed between, e.g., an epitope and an MHC polypeptide;between an MHC polypeptide and an immunomodulatory polypeptide; betweenan MHC polypeptide and an Ig Fc polypeptide; between a first variantIL-2 polypeptide and a second variant IL-2 polypeptide; or a between asecond variant IL-2 polypeptide and a third variant IL-2 polypeptide.

Suitable linkers (also referred to as “spacers”) can be readily selectedand can be of any of a number of suitable lengths, such as from 1 aminoacid to 25 amino acids, from 3 amino acids to 20 amino acids, from 2amino acids to 15 amino acids, from 3 amino acids to 12 amino acids,including 4 amino acids to 10 amino acids, 5 amino acids to 9 aminoacids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 aminoacids. A suitable linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids inlength.

Exemplary linkers include glycine polymers (G), glycine-serine polymers(including, for example, (GS)_(n), (GSGGS)_(n) (SEQ ID NO:89) and(GGGS)_(n) (SEQ ID NO:86), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art, Glycine and glycine-serine polymers can beused; both Gly and Ser are relatively unstructured, and therefore canserve as a neutral tether between components, Glycine polymers can beused; glycine accesses significantly more phi-psi space than evenalanine, and is much less restricted than residues with longer sidechains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary linkers can comprise amino acid sequences including, but notlimited to, GGSG (SEQ ID NO:2), GGSGG (SEQ ID NO:3), GSGSG (SEQ IDNO:4), GSGGG (SEQ ID NO:5), GGGSG (SEQ ID NO:6), GSSSG (SEQ ID NO:7),and the like. Exemplary linkers can include, e.g., Gly(Ser₄)n, where nis 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, a linker comprisesthe amino acid sequence (GSSSS)n (SEQ ID NO:93), where n is 4. In somecases, a linker comprises the amino acid sequence (GSSSS)n (SEQ IDNO:94), where n is 5. In some cases, a linker comprises the amino acidsequence (GGGGS)n (SEQ ID NO:9), where n is 1. In some cases, a linkercomprises the amino acid sequence (GGGCS)n (SEQ ID NO:92), where n is 2.In some cases, a linker comprises the amino acid sequence (GGGS)n (SEQID NO:89), where n is 3. In some cases, a linker comprises the aminoacid sequence (GGGGS)n (SEQ ID NO:90), where n is 4. In some cases, alinker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:91), wheren is 5.

In some cases, a linker polypeptide, present in a first polypeptide of amultimeric polypeptide of the present disclosure, includes a cysteineresidue that can form a disulfide bond with a cysteine residue presentin a second polypeptide of a multimeric polypeptide of the presentdisclosure. In some cases, for example, a suitable linker comprises theamino acid sequence GCASGGGGSGGGGS (SEQ ID NO:10).

Epitopes

An epitope present in a multimeric polypeptide of the present disclosurecan have a length of from about 4 amino acids to about 25 amino acids,e.g., the epitope can have a length of from 4 amino acids (aa) to 10 aa,from 10 aa to 15 aa, from 15 as to 20 aa, or from 20 aa to 25 aa. Forexample, an epitope present in a multimeric polypeptide of the presentdisclosure can have a length of 4 amino acids (aa), 5 an, 6 an, 7, an, 8an, 9 an, 10 aa, 11 an, 12 as, 13 an, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa,19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 an. In some cases, anepitope present in a multimeric polypeptide of the present disclosurehas a length of from 5 amino acids to 10 amino acids, e.g., 5 aa, 6 aa,7 aa, 8 aa, 9 aa, or 10 aa.

An epitope present in a multimeric polypeptide of the present disclosureis specifically bound by a T-cell, i.e., the epitope is specificallybound by an epitope-specific T cell. An epitope-specific T cell hinds anepitope having a reference amino acid sequence, but does notsubstantially bind an epitope that differs from the reference amino acidsequence. For example, an epitope-specific T cell binds an epitopehaving a reference amino acid sequence, and binds an epitope thatdiffers from the reference amino acid sequence, if at all, with anaffinity that is less than 10⁶ M, less than 10⁵ M, or less than 10⁴ M.An epitope-specific T cell can bind an epitope for which it is specificwith an affinity of at least 10⁷ M, at least 10⁸ M, at least 10⁹ M, orat least 10¹⁰ M.

Suitable epitopes include, but are not limited to, epitopes present in acancer-associated antigen. Cancer-associated antigens include, but arenot limited to, α-folate receptor; carbonic anhydrase IX (CAIX); CD19;CD20; CD22; CD30; CD33; CD44v7/8; carcinoembryonic antigen (CEA);epithelial glycoprotein-2 (EGP-2); epithelial glycoprotein-40 (EGP-40);folate binding protein (FBP); fetal acetylcholine receptor, gangliosideantigen GD2; Her2/ncu; IL-13R-a2; kappa light chain; LeY; L1 celladhesion molecule; melanoma-associated antigen (MAGE); MAGE-A1;mesothelin; MUC1; NKG2D ligands; oncofetal antigen (h5T4); prostate stemcell antigen (PSCA); prostate-specific membrane antigen (PSMA);rumor-associate glycoprotein-72 (TAG-72); and vascular endothelialgrowth factor receptor-2 (VEGF-R2). See, e.g., Vigneron et al. (2013)Cancer Immunity 13:15; and Vigneron (2015) BioMed Res. Int'l Article ID948501. In some cases, the epitope is a human papilloma virus E7 antigenepitope; see, e.g., Ramos et al. (2013) J. Immunother. 36:66.

In some cases, the epitope is HPV16E7/82-90 (LLMGTLGIV; SEQ ID NO:11).In some cases, the epitope is HPV16E7/86-93 (TLGIVCPI; SEQ ID NO:12). Insome cases, the epitope is HPV16E7/11-20 (YMLDLQPETT; SEQ ID NO:13). Insome cases, the epitope is HPV16E7/11-19 (YMLDLQPET; SEQ ID NO:87). See,e.g., Ressing et al. ((1995) J. Immunol. 154:5934) for additionalsuitable HPV epitopes.

MHC Polypeptides

As noted above, a multimeric polypeptide of the present disclosureincludes MHC polypeptides. For the purposes of the instant disclosure,the term “major histocompatibility complex (MHC) polypeptides” is meantto include MHC polypeptides of various species, including human MHC(also referred to as human leukocyte antigen (HLA)) polypeptides, rodent(e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides of othermammalian species (e.g., lagomorphs, non-human primates, canines,felines, ungulates (e.g., equines, bovines, ovines, caprines, etc.), andthe like. The term “MHC polypeptide” is meant to include Class I MHCpolypeptides (e.g., β-2 microglobulin and MHC class I heavy chain) andMHC Class II polypeptides (e.g., MHC Class II α polypeptide and MHCClass II 1 polypeptide).

As noted above, in some embodiments of a multimeric polypeptide of thepresent disclosure, the first and the second MHC polypeptides are ClassI MHC polypeptides; e.g., in some cases, the first MHC polypeptide is anMHC Class I β2-microglobulin (β2M) polypeptide, and the second MHCpolypeptide is an MHC Class I heavy chain (H chain). In other cases, thefirst and the second MHC polypeptides are Class II MHC polypeptides;e.g., in some cases, the first MHC polypeptide is an MHC Class IIα-chain polypeptide, and the second MHC polypeptide is an MHC Class IIβ-chain polypeptide. In other cases, the first polypeptide is an MHCClass II β-chain polypeptide, and the second MHC polypeptide is an MHCClass II α-chain polypeptide.

In some cases, an MHC polypeptide of a multimeric polypeptide of thepresent disclosure is a human MHC polypeptide, where human MHCpolypeptides are also referred to as “human leukocyte antigen” (“HLA”)polypeptides. In some cases, an MHC polypeptide of a multimericpolypeptide of the present disclosure is a Class I HLA polypeptide,e.g., a β2-microglobulin polypeptide, or a Class I HLA heavy chainpolypeptide. Class I HLA heavy chain polypeptides include HLA-A heavychain polypeptides, HLA-B heavy chain polypeptides, HLA-C heavy chainpolypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chainpolypeptides, and HLA-G heavy chain polypeptides. In some cases, an MHCpolypeptide of a multimeric polypeptide of the present disclosure is aClass II HLA polypeptide, e.g., a Class II HLA α chain or a Class II HLAα chain, MHC Class 11 polypeptides include MCH Class 11 DP α and βpolypeptides, DM α and β polypeptides, DOA α and β polypeptides, DOB αand β polypeptides, DQ α and β polypeptides, and DR α and βpolypeptides.

As an example, an MHC Class I heavy chain polypeptide of a multimericpolypeptide of the present disclosure can comprise an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to amino acids 25-365 of the amino acid sequence of the humanHLA-A heavy chain polypeptide depicted in FIG. 5A.

As an example, an MHC Class I heavy chain polypeptide of a multimericpolypeptide of the present disclosure can comprise an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to amino acids 25-365 of the amino acid sequence of thefollowing human HLA-A heavy chain amino acid sequence:

(SEQ ID NO: 14) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP.

As another example, an MHC Class I heavy chain polypeptide of amultimeric polypeptide of the present disclosure can comprise an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to amino acids 25-362 of the amino acid sequence ofthe human HLA-B heavy chain polypeptide depicted in FIG. 5B.

As another example, an MHC Class I heavy chain polypeptide of amultimeric polypeptide of the present disclosure can comprise an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to amino acids 25-362 of the amino acid sequence ofthe human HLA-C heavy chain polypeptide depicted in FIG. 5C.

As another example, an MHC Class I heavy chain polypeptide of amultimeric polypeptide of the present disclosure can comprise an aminoacid sequence having at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the following amino acid sequence:

(SEQ ID NO: 15) GPHSLRYFVTAVSRPGLGEPRFIAVGYVDDTQFVRFDSDADNPRFEPRAPWMEQEGPEYWEEQTQRAKSDEQWFRVSLRTAQRYYNQSKGGSHTFQRMFGCDVGSDWRLLRGYQQFAYDGRDYIALNEDLKTWTAADTAALITRRKWEQAGDAEYYRAYLEGECVEWLRRYLELGNETLLRTDSPKAHVTYHPRSQVDVTLRCWALGFYPADITLTWQLNGEDLTQDMELVETRPAGDGTFQKWAAVVVPLGKEQNYTCHVHHKGLPEPLTLRW.

A β2-microglobulin (β2M) polypeptide of a multimeric polypeptide of thepresent disclosure can be a human β2M polypeptide, a non-human primateβ2M polypeptide, a murine β2M polypeptide, and the like. In someinstances, a β2M polypeptide comprises an amino acid sequence having atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100%, amino acid sequence identity to a β2Mamino acid sequence depicted in FIG. 6. In some instances, a β2Mpolypeptide comprises an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100%, amino acid sequence identity to amino acids 21 to119 of a β2M amino acid sequence depicted in FIG. 6.

In some cases, an MHC polypeptide comprises a single amino acidsubstitution relative to a reference MHC polypeptide (where a referenceMHC polypeptide can be a wild-type MHC polypeptide), where the singleamino acid substitution substitutes an amino acid with a cysteine (Cys)residue. Such cysteine residues, when present in an MHC polypeptide of afirst polypeptide of a multimeric polypeptide of the present disclosure,can form a disulfide bond with a cysteine residue present in a secondpolypeptide chain of a multimeric polypeptide of the present disclosure.

In some cases, a first MHC polypeptide in a first polypeptide of amultimeric polypeptide of the present disclosure, and/or the second MHCpolypeptide in the second polypeptide of a multimeric polypeptide of thepresent disclosure, includes an amino acid substitution to substitute anamino acid with a cysteine, where the substituted cysteine in the firstMHC polypeptide forms a disulfide bond with a cysteine in the second MHCpolypeptide, where a cysteine in the first MHC polypeptide forms adisulfide bond with the substituted cysteine in the second MHCpolypeptide, or where the substituted cysteine in the first MHCpolypeptide forms a disulfide bond with the substituted cysteine in thesecond MHC polypeptide.

For example, in some cases, one of following pairs of residues in an HLAβ2-microglobulin and an HLA Class I heavy chain is substituted withcysteines (where residue numbers are those of the maturepolypeptide): 1) β2M residue 12, HLA Class I heavy chain residue 236; 2)β2M residue 12, HLA Class I heavy chain residue 237; 3) β2M residue 8,HLA Class I heavy chain residue 234; 4) β2M residue 10, HLA Class Iheavy chain residue 235; 5) β2M residue 24, HLA Class I heavy chainresidue 236; 6) β2M residue 28, HLA Class I heavy chain residue 232; 7)β2M residue 98, HLA Class I heavy chain residue 192: 8) β2M residue 99,HLA Class I heavy chain residue 234; 9) β2M residue 3, HLA Class I heavychain residue 120; 10) β2M residue 31, HLA Class I heavy chain residue96; 11) β2M residue 53, HLA Class I heavy chain residue 35; 12) β2Mresidue 60, HLA Class I heavy chain residue 96; 13) β2M residue 60, HLAClass I heavy chain residue 122; 14) β2M residue 63, HLA Class I heavychain residue 27; 15) β2M residue Arg3, HLA Class I heavy chain residueGly120: 16) (2M residue His31, HLA Class I heavy chain residue Gln96;17) 12M residue Asp53, HLA Class I heavy chain residue Arg35; 18) β2Mresidue Trp60, HLA Class I heavy chain residue Gln96; 19) β2M residueTrp60, HLA Class I heavy chain residue Asp122; 20) (β2M residue Tyr63,HLA Class I heavy chain residue Tyr27; 21) β2M residue Lys6, HLA Class Iheavy chain residue Glu232: 22) β2M residue Gln8, HLA Class I heavychain residue Arg234: 23) β2M residue Tyr10, HLA Class I heavy chainresidue Pro235; 24) β2M residue Ser11, HLA Class I heavy chain residueGln242; 25) β2M residue Asn24, HLA Class I heavy chain residue Ala236;26) β2M residue Ser28, HLA Class I heavy chain residue Glu232; 27) β2Mresidue Asp98, HLA Class I heavy chain residue His192; and 28) β2Mresidue Met99, HLA Class I heavy chain residue Arg234. The amino acidnumbering of the MHC/HLA Class I heavy chain is in reference to themature MHC/HLA Class I heavy chain, without a signal peptide. Forexample, in the amino acid sequence depicted in FIG. 5A, which includesa signal peptide, Gly120 is Gly144; Gln96 is Gln120; etc. In some cases,the β2M polypeptide comprises an R12C substitution, and the HLA Class Iheavy chain comprises an A236C substitution; in such cases, a disulfidebond forms between Cys-12 of the β2M polypeptide and Cys-236 of the HLAClass I heavy chain. For example, in some cases, residue 236 of themature HLA-A amino acid sequence (i.e., residue 260 of the amino acidsequence depicted in FIG. 5A) is substituted with a Cys. In some cases,residue 236 of the mature HLA-B amino acid sequence (i.e., residue 260of the amino acid sequence depicted in FIG. 5B) is substituted with aCys. In some cases, residue 236 of the mature HLA-C amino acid sequence(i.e., residue 260 of the amino acid sequence depicted in FIG. 5C) issubstituted with a Cys. In some cases, residue 32 (corresponding toArg-12 of mature β2M) of an amino acid sequence depicted in FIG. 6 issubstituted with a Cys.

In some cases, a β2M polypeptide comprises the amino acid sequence:IQRTPKIQVY SRHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID) NO:16). In somecases, a β2M polypeptide comprises the amino acid sequence: IQRTPKIQVYSCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEFTPTEKDEYAC RVNHIVTLSQP KIVKWDRDM (SEQ ID NO:17).

In some cases, an HLA Class I heavy chain polypeptide comprises theamino acid sequence:

(SEQ ID NO: 14) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDCKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP AGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP.

In some cases, an HLA Class I heavy chain polypeptide comprises theamino acid sequence:

(SEQ ID NO: 18) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP CGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP.

In some cases, an HLA Class I heavy chain polypeptide comprises theamino acid sequence:

(SEQ ID NO: 19) GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP CGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE.

In some cases, the fβ2M polypeptide comprises the following amino acidsequence:

IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:17); and the HLAClassI heavy chain polypeptide of a multimeric polypeptide of thepresent disclosure comprises the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRVYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDCEDQTQDTELVETRPCCDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:18), where the Cys residues thatare underlined and in bold form a disulfide bond with one another in themultimeric polypeptide.

In some cases, the β2M polypeptide comprises the amino acid sequence:

(SEQ ID NO: 17) IQRTPKIQVYS C HPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVK WDRDM.

Immunomodulatory Polypeptides

A multimeric polypeptide of the present disclosure comprises a variantIL-2 polypeptide of the present disclosure, as described above, that isa variant of a naturally occurring costimulatory protein, which variantexhibits a reduced affinity for its counterpart (cognate) costimulatoryprotein on the T cell (e.g., IL-2R) as compared to the affinity of thenaturally occurring IL-2 polypeptide for the counterpart costimulatoryprotein (IL-2R). Thus, a multimeric polypeptide of the presentdisclosure comprises the variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure.

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2E,where amino acid 16 is an amino acid other than a histidine, e.g., whereamino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr,Cys, Met. Asn, Gln, Lys, Arg. Asp, or Glu. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2E, where amino acid 16 isAla, Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2E, where aminoacid 16 is Gly. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2E, where amino acid 16 is Val. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2E, where amino acid 16 isLeu. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2E,where amino acid 16 is Ile. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Asn. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2E, where aminoacid 16 is Asp. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2E, where amino acid 16 is Cys. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2E, where amino acid 16 isGln. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2E,where amino acid 16 is Glu. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Met. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2E, where aminoacid 16 is Phe. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2E, where amino acid 16 is Ser. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2E, where amino acid 16 isThr. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2E,where amino acid 16 is Trp. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2E, where amino acid 16 is Tyr. In some cases, thevariant IL-2 polypeptide has a binding affinity for IL-2R that is fromabout 100 nM to 150 nM, from about 150 nM to about 200 nM, from about200 nM to about 250 nM, from about 250 nM to about 300 nM, from about300 nM to about 350 nM, from about 350 nM to about 400 nM, from about400 nM to about 500 nM, from about 500 nM to about 600 nM, from about600 nM to about 700 nM, from about 700 nM to about 800 nM, from about800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μMto about 5 μM, from about 5 μM to about 10 μM, from about 10 M to about15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM,from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, orfrom about 75 μM to about 100 μM. In some cases, the variant IL-2polypeptide has a length of 133 amino acids.

F42 Substitution

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2B,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2B, where amino acid 42 isAla, Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2B, where amino acid 42 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2B, where aminoacid 42 is Gly. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2B, where amino acid 42 is Val. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2B, where amino acid 42 isLeu. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2B,where amino acid 42 is Ile. In some cases, a single copy of the variantIL-2 polypeptide is present in a multimeric polypeptide of the presentdisclosure. In some cases, a multimeric polypeptide of the presentdisclosure comprises two copies of the variant IL-2 polypeptide, e.g.,where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the (β2M polypeptide. In some cases, the variant IL-2polypeptide, or the synTac comprising same, has a binding affinity forIL-2R that is from about 100 nM to 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 500 nM, from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, fromabout 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

Y45 Substitution

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2F,where amino acid 45 is an amino acid other than a tyrosine, e.g., whereamino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys,Met. Asn, Gln, Lys, Arg, His, Asp, or Glu. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2F, where amino acid 45 isAla, Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2F, where amino acid 45 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2F, where aminoacid 45 is Gly. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2F, where amino acid 45 is Val. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2F, where amino acid 45 isLeu. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2F,where amino acid 45 is Ile. In some cases, a single copy of the variantIL-2 polypeptide is present in a multimeric polypeptide of the presentdisclosure. In some cases, a multimeric polypeptide of the presentdisclosure comprises two copies of the variant IL-2 polypeptide, e.g.,where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/arc on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or the synTac comprising same, has a binding affinity forIL-2R that is from about 100 nM to 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 500 nM, from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, fromabout 10 M to about 15 M, from about 15 μM to about 20 μM, from about 20μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM toabout 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

Q126 Substitution

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2G,where amino acid 126 is an amino acid other than a glutamine, e.g.,where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Lys, Arg, His. Asp, or Glu. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2G, where aminoacid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2G, where amino acid 126 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2G,where amino acid 126 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2G, where amino acid 126 is Val. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2G, where aminoacid 126 is Leu. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2G, where amino acid 126 is ile. In some cases, a single copy ofthe variant IL-2 polypeptide is present in a multimeric polypeptide ofthe present disclosure. In some cases, a multimeric polypeptide of thepresent disclosure comprises two copies of the variant IL-2 polypeptide,e.g., where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand D2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand 132M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL-2R that is from about 100 nM to 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 500 nM, from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, fromabout 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42 and H16 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2H,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; and where amino acid 16is an amino acid other than a histidine, e.g., where amino acid 16 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met. Asn,Gln, Lys, Arg, Asp, or Glu. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2H, where amino acid 42 is Ala, Gly, Val, Leu, or Ile;and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2H, where aminoacid 42 is Ala and amino acid 16 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2H, where amino acid 42 isAla and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2H, where amino acid 42 is Val and amino acid 16 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2H,where amino acid 42 is Leu, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2H, where aminoacid 42 is Ile and amino acid 16 is Ala. In some cases, a single copy ofthe variant IL-2 polypeptide is present in a multimeric polypeptide ofthe present disclosure. In some cases, a multimeric polypeptide of thepresent disclosure comprises two copies of the variant IL-2 polypeptide,e.g., where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand fβ2M, the IL-2 polypeptide(s) is are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the D2M polypeptide. In some cases, a multimeric polypeptideof the present disclosure comprises 2 copies of the IL-2 variantcomprising F42A and H16A substitutions, where the multimeric polypeptidecomprises HLA Class I heavy chain and β2M polypeptides, and where the 2copies of IL-2 (F42A, H16A) are on the polypeptide chain comprising theHLA Class I heavy chain. In some cases, the variant IL-2 polypeptide, ora synTac comprising same, has a binding affinity for IL-2R that is fromabout 100 nM to 150 nM, from about 150 nM to about 200 nM, from about200 nM to about 250 nM, from about 250 nM to about 300 nM, from about300 nM to about 350 nM, from about 350 nM to about 400 nM, from about400 nM to about 500 nM, from about 500 nM to about 600 nM, from about600 nM to about 700 nM, from about 700 nM to about 800 nM, from about800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μMto about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM,from about 25 μM to about 50 μM, from about 50 μM to about 75 M, or fromabout 75 μM to about 100 μM. In some cases, the variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure has alength of 133 amino acids. In some cases, the variant IL-2 polypeptidecomprises the amino acid sequence depicted in FIG. 34B (comprising H16Aand F42A substitutions).

F42 and D20 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 21,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; and where amino acid 20is an amino acid other than an aspartic acid, e.g., where amino acid 20is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met. Asn,Gln, Lys, Arg, His, or Glu. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 21, where amino acid 42 is Ala, Gly, Val, Leu, or Ile;and where amino acid 20 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 21, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; and where amino acid 20 is Asn,Gln, Lys, Arg, or His. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 21, where amino acid 42 is Ala and amino acid 20 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2I,where amino acid 42 is Ala and amino acid 20 is Gly. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2I, where aminoacid 42 is Val and amino acid 20 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2I, where amino acid 42 isLeu, and amino acid 20 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2I, where amino acid 42 is Ile and amino acid 20 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2I,where amino acid 42 is Ala and amino acid 20 is Asn. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2I, where aminoacid 42 is Ala and amino acid 20 is Gln. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2I, where amino acid 42 isAla and amino acid 20 is Lys. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2I, where amino acid 42 is Ala and amino acid 20 isArg. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2I,where amino acid 42 is Ala and amino acid 20 is His. In some cases, asingle copy of the variant IL-2 polypeptide is present in a multimericpolypeptide of the present disclosure. In some cases, a multimericpolypeptide of the present disclosure comprises two copies of thevariant IL-2 polypeptide, e.g., where the two copies are in tandem withno linker between the two copies, or are in tandem and separated by alinker peptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises three copies of the variant IL-2 polypeptide, e.g.,where the three copies are in tandem with no linker between the threecopies, or are in tandem and separated by a linker peptide. In somecases, where an IL-2synTac of the present disclosure comprises HLA ClassI heavy chain and 132M, the IL-2 polypeptide(s) is/are on thepolypeptide chain comprising the HLA Class I heavy chain. In some cases,where an IL-2/synTac of the present disclosure comprises HLA Class Iheavy chain and Jβ2M, the IL-2 polypeptide(s) is/are on the polypeptidechain comprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL-2R that is from about 100 nM to 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 500 nM, from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, fromabout 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, D20, and E15 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His. Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met. Asn,Gln, Lys, Arg, His, or Glu; and where amino acid 15 is an amino acidother than a glutamic acid, e.g., where amino acid 15 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg,His, or Asp. In some cases, a variant IL-2 polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2I, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu,or Ile; and where amino acid 15 is Ala, Gly, Val, Leu, or Ile. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2I, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln,Lys, Arg, or His; and where amino acid 15 is Ala, Gly, Val, Leu, or Ile.In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 15 isGly. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Val, amino acid 20 is Ala, and amino acid 15 isGly. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2I,where amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 15 isGly. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2I,where amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2J,where amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 15 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2I,where amino acid 42 is Ala, amino acid 20 is His, and amino acid 15 isAla. In some cases, a single copy of the variant IL-2 polypeptide ispresent in a multimeric polypeptide of the present disclosure. In somecases, a multimeric polypeptide of the present disclosure comprises twocopies of the variant IL-2 polypeptide, e.g., where the two copies arein tandem with no linker between the two copies, or are in tandem andseparated by a linker peptide. In some cases, where an IL-2/synTac ofthe present disclosure comprises HLA Class I heavy chain and β2M, theIL-2 polypeptide(s) is/arc on the polypeptide chain comprising the HLAClass I heavy chain. In some cases, where an IL-2/synTac of the presentdisclosure comprises HLA Class I heavy chain and β2M, the IL-2polypeptide(s) is/are on the polypeptide chain comprising the β2Mpolypeptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises three copies of the variant IL-2 polypeptide, e.g.,where the three copies are in tandem with no linker between the threecopies, or are in tandem and separated by a linker peptide. In somecases, the variant IL-2 polypeptide, or a synTac comprising same, has abinding affinity for IL-2R that is from about 100 nM to 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 500 nM, fromabout 500 nM to about 600 nM, from about 600 nM to about 700 nM, fromabout 700 nM to about 800 nM, from about 800 nM to about 900 nM, fromabout 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μMto about 10 μM, from about 10 μM to about 15 μM, from about 15 μM toabout 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100μM. In some cases, the variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure has a length of 133 amino acids.

F42, D20, and 1116 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2K,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met. Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; and where amino acid 16 is an amino acidother than a histidine, e.g., where amino acid 16 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met. Asn, Gln, Lys, Arg, Asp, orGlu. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2K,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2K, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where aminoacid 20 is Asn, Gln, Lys, Arg, or His; and where amino acid 16 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Gly,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Val, amino acid 20 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Leu, amino acid 20 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ile, amino acid 20 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Asn,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Gln,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Lys,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is Arg,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2K, where amino acid 42 is Ala, amino acid 20 is His,and amino acid 16 is Ala. In some cases, a single copy of the variantIL-2 polypeptide is present in a multimeric polypeptide of the presentdisclosure. In some cases, a multimeric polypeptide of the presentdisclosure comprises two copies of the variant IL-2 polypeptide, e.g.,where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand fβ2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL-2R that is from about 100 nM to 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 500 nM, from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, fromabout 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, D20, and Q126 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2L,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; and where amino acid 126 is an amino acidother than a glutamine, e.g., where amino acid 126 is Gly, Ala, Val,Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His,Asp, or Glu. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2L, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where aminoacid 20 is Ala, Gly, Val, Leu, or Ile; and where amino acid 126 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2L, where amino acid 42 is Ala, Gly, Val, Leu, or Ile;where amino acid 20 is Asn, Gln, Lys, Arg, or His; and where amino acid126 is Ala, Gly, Val, Leu, or Ile. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2L, where amino acid 42 isAla, amino acid 20 is Ala, and amino acid 126 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Gly, and amino acid 126 is Gly. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Val, amino acid 20 is Ala, and amino acid 126 is Gly. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Leu, amino acid 20 is Ala, and amino acid 126 is Gly. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ile, amino acid 20 is Ala, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Asn, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Gln, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Lys, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is Arg, and amino acid 126 is Ala. In somecases, a variant IL-2 polypeptide present in a multimeric polypeptide ofthe present disclosure comprises an amino acid sequence having at least90%, at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2L, where aminoacid 42 is Ala, amino acid 20 is His, and amino acid 126 is Ala. In somecases, a single copy of the variant IL-2 polypeptide is present in amultimeric polypeptide of the present disclosure. In some cases, amultimeric polypeptide of the present disclosure comprises two copies ofthe variant IL-2 polypeptide, e.g., where the two copies are in tandemwith no linker between the two copies, or are in tandem and separated bya linker peptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises three copies of the variant IL-2 polypeptide, e.g.,where the three copies are in tandem with no linker between the threecopies, or are in tandem and separated by a linker peptide. In somecases, where an IL-2/synTac of the present disclosure comprises HLAClass I heavy chain and β2M, the IL-2 polypeptide(s) is/are on thepolypeptide chain comprising the HLA Class I heavy chain. In some cases,where an IL-2/synTac of the present disclosure comprises HLA Class Iheavy chain and β2M, the IL-2 polypeptide(s) is/are on the polypeptidechain comprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL-2R that is from about 100 nM to 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 500 nM, from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, fromabout 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, or from about 75 M to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, D20, and Y45 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2M,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; and where amino acid 45 is an amino acidother than a tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu,Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His. Asp, orGlu. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2M,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20is Ala, Gly, Val, Leu, or Ile; and where amino acid 45 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2M, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where aminoacid 20 is Asn, Gln, Lys, Arg, or His; and where amino acid 45 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Ala,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Gly,and amino acid 45 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Val, amino acid 20 is Ala,and amino acid 45 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Leu, amino acid 20 is Ala,and amino acid 45 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ile, amino acid 20 is Ala,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Asn,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Gln,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Lys,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is Arg,and amino acid 45 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2M, where amino acid 42 is Ala, amino acid 20 is His,and amino acid 45 is Ala. In some cases, a single copy of the variantIL-2 polypeptide is present in a multimeric polypeptide of the presentdisclosure. In some cases, a multimeric polypeptide of the presentdisclosure comprises two copies of the variant IL-2 polypeptide, e.g.,where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the 132M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL-2R that is from about 100 nM to 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 500 nM, from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1μM, to about 1 μM to about 5 M, from about 5 μM to about 10 μM, fromabout 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, D20, Y45, and H16 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2N,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met. Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; where amino acid 45 is an amino acid otherthan a tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu;and where amino acid 16 is an amino acid other than a histidine, e.g.,where amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser,Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu,or Ile; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; and whereamino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln, Lys, Arg,or His; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; and whereamino acid 16 is Ala, Gly, Val, Leu, or Ile. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 16 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2N,where amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2N, where amino acid 42 is Val, amino acid 20 is Ala,amino acid 45 is Gly, and amino acid 16 is Ala. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isLeu, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 16 isVal. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2N,where amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2N, where amino acid 42 is Ala, amino acid 20 is Asn,amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, amino acid 20 is Gln, amino acid 45 is Ala, and amino acid 16 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2N,where amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2N, where amino acid 42 is Ala, amino acid 20 is Arg,amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, a variantIL-2 polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2N, where amino acid 42 isAla, amino acid 20 is His, amino acid 45 is Ala, and amino acid 16 isAla. In some cases, a single copy of the variant IL-2 polypeptide ispresent in a multimeric polypeptide of the present disclosure. In somecases, a multimeric polypeptide of the present disclosure comprises twocopies of the variant IL-2 polypeptide, e.g., where the two copies arein tandem with no linker between the two copies, or are in tandem andseparated by a linker peptide. In some cases, a multimeric polypeptideof the present disclosure comprises three copies of the variant IL-2polypeptide, e.g., where the three copies are in tandem with no linkerbetween the three copies, or are in tandem and separated by a linkerpeptide. In some cases, where an IL-2/synTac of the present disclosurecomprises HLA Class I heavy chain and 12M, the IL-2 polypeptide(s)is/are on the polypeptide chain comprising the HLA Class I heavy chain.In some cases, where an IL-2/synTac of the present disclosure comprisesHLA Class I heavy chain and β2M, the IL-2 polypeptide(s) is/are on thepolypeptide chain comprising the β2M polypeptide. In some cases, thevariant IL-2 polypeptide, or a synTac comprising same, has a bindingaffinity for IL-2R that is from about 100 nM to 150 nM, from about 150nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nMto about 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 500 nM, from about 500 nM toabout 600 nM, from about 600 nM to about 700 nM, from about 700 nM toabout 800 nM, from about 800 nM to about 900 nM, from about 900 nM toabout 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM,from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, fromabout 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about50 μM to about 75 μM, or from about 75 μM to about 100 (M. In somecases, the variant IL-2 polypeptide present in a multimeric polypeptideof the present disclosure has a length of 133 amino acids.

F42, D20, Y45, and Q126 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 20,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met. Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; where amino acid 45 is an amino acid otherthan a tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu;and where amino acid 126 is an amino acid other than a glutamine, e.g.,where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Pro, Tyr, Trp,Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp, or Glu. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 20, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly,Val, Leu, or Ile; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; andwhere amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 20, where aminoacid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 20 is Asn, Gln,Lys, Arg, or His; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; andwhere amino acid 126 is Ala, Gly, Val, Leu, or Ile. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 20, where aminoacid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 20, where amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45is Gly, and amino acid 126 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 20, where amino acid 42 isVal, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 126 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 20,where amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly,and amino acid 126 is Val. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 20, where amino acid 42 is Ile, amino acid 20 is Ala,amino acid 45 is Ala, and amino acid 126 is Gly. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 20, where aminoacid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and aminoacid 126 is Ala. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 20, where amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45is Ala, and amino acid 126 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 20, where amino acid 42 isAla, amino acid 20 is Lys, amino acid 45 is Ala, and amino acid 126 isAla. In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 20,where amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala,and amino acid 126 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 20, where amino acid 42 is Ala, amino acid 20 is His,amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, a singlecopy of the variant IL-2 polypeptide is present in a multimericpolypeptide of the present disclosure. In some cases, a multimericpolypeptide of the present disclosure comprises two copies of thevariant IL-2 polypeptide, e.g., where the two copies are in tandem withno linker between the two copies, or are in tandem and separated by alinker peptide. In some cases, a multimeric polypeptide of the presentdisclosure comprises three copies of the variant IL-2 polypeptide, e.g.,where the three copies are in tandem with no linker between the threecopies, or are in tandem and separated by a linker peptide. In somecases, where an IL-2/synTac of the present disclosure comprises HLAClass I heavy chain and D2M, the IL-2 polypeptide(s) is/are on thepolypeptide chain comprising the HLA Class I heavy chain. In some cases,where an IL-2/synTac of the present disclosure comprises HLA Class Iheavy chain and (β2M, the IL-2 polypeptide(s) is/are on the polypeptidechain comprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising sane, has a binding affinity forIL-2R that is from about 100 nM to 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 500 nM, from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, fromabout 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about20 μM to about 25 LAM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, D20, Y45, H16, and Q126 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu; where amino acid 20 isan amino acid other than an aspartic acid, e.g., where amino acid 20 isGly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn,Gln, Lys, Arg, His, or Glu; where amino acid 45 is an amino acid otherthan a tyrosine, e.g., where amino acid 45 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Glu;where amino acid 126 is an amino acid other than a glutamine, e.g.,where amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp,Ser, Thr, Cys, Met. Asn, Lys, Arg, His. Asp, or Glu; and where aminoacid 16 is an amino acid other than a histidine, e.g., where amino acid16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met.Asn, Gln, Lys, Arg. Asp, or Glu. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2P, where amino acid 42 isAla, Gly, Val, Leu, or Ile; where amino acid 20 is Ala, Gly, Val, Leu,or Ile; where amino acid 45 is Ala, Gly, Val, Leu, or Ile; where aminoacid 126 is Ala, Gly, Val, Leu, or Ile; and where amino acid 16 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2P, where amino acid 42 is Ala, Gly, Val, Leu, or Ile;where amino acid 20 is Asn, Gln, Lys, Arg, or His; where amino acid 45is Ala, Gly, Val, Leu, or Ile; where amino acid 126 is Ala, Gly, Val,Leu, or Ile; and where amino acid 16 is Ala, Gly, Val, Leu, or Ile. Insome cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala,amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2P, where aminoacid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, amino acid126 is Ala, and amino acid 16 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2P, where amino acid 42 isVal, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2P, where amino acid 42 is Leu, amino acid 20 is Ala,amino acid 45 is Gly, amino acid 126 is Val, and amino acid 16 is Ala.In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala,amino acid 126 is Gly, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2P, where aminoacid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, amino acid126 is Ala, and amino acid 16 is Ala. In some cases, a variant IL-2polypeptide present in a multimeric polypeptide of the presentdisclosure comprises an amino acid sequence having at least 90%, atleast 95%, at least 98%, or at least 99%, amino acid sequence identityto the amino acid sequence depicted in FIG. 2P, where amino acid 42 isAla, amino acid 20 is Gln, amino acid 45 is Ala, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2P, where amino acid 42 is Ala, amino acid 20 is Lys,amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala.In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2P,where amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala,amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, avariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure comprises an amino acid sequence having at least 90%,at least 95%, at least 98%, or at least 99%, amino acid sequenceidentity to the amino acid sequence depicted in FIG. 2P, where aminoacid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, amino acid126 is Ala, and amino acid 16 is Ala. In some cases, a single copy ofthe variant IL-2 polypeptide is present in a multimeric polypeptide ofthe present disclosure. In some cases, a multimeric polypeptide of thepresent disclosure comprises two copies of the variant IL-2 polypeptide,e.g., where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand β2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising same, has a binding affinity forIL-2R that is from about 100 nM to 150 nM, from about 150 nM to about200 nM, from about 200 nM to about 250 nM, from about 250 nM to about300 nM, from about 300 nM to about 350 nM, from about 350 nM to about400 nM, from about 400 nM to about 500 nM, from about 500 nM to about600 nM, from about 600 nM to about 700 nM, from about 700 nM to about800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, fromabout 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μMto about 75 μM, or from about 75 μM to about 100 μM. In some cases, thevariant IL-2 polypeptide present in a multimeric polypeptide of thepresent disclosure has a length of 133 amino acids.

F42, Q126, and H16 Substitutions

In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2Q,where amino acid 42 is an amino acid other than a phenylalanine, e.g.,where amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr,Cys, Met, Asn, Gln, Lys, Arg, His, Asp, or Gln; where amino acid 126 isan amino acid other than a glutamine, e.g., where amino acid 126 is Gly,Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met. Asn, Lys,Arg, His, Asp, or Glu; and where amino acid 16 is an amino acid otherthan a histidine, e.g., where amino acid 16 is Gly, Ala, Val, Leu, Ile,Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp, or Glu.In some cases, a variant IL-2 polypeptide present in a multimericpolypeptide of the present disclosure comprises an amino acid sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%, aminoacid sequence identity to the amino acid sequence depicted in FIG. 2Q,where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where amino acid 126is Ala, Gly, Val, Leu, or lie; and where amino acid 16 is Ala, Gly, Val,Leu, or Ile. In some cases, a variant IL-2 polypeptide present in amultimeric polypeptide of the present disclosure comprises an amino acidsequence having at least 90%, at least 95%, at least 98%, or at least99%, amino acid sequence identity to the amino acid sequence depicted inFIG. 2Q, where amino acid 42 is Ala, Gly, Val, Leu, or Ile; where aminoacid 126 is Asn, Gln, Lys, Arg, or His; and where amino acid 16 is Ala,Gly, Val, Leu, or Ile. In some cases, a variant IL-2 polypeptide presentin a multimeric polypeptide of the present disclosure comprises an aminoacid sequence having at least 90%, at least 95%, at least 98%, or atleast 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Gly,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Val, amino acid 126 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Leu, amino acid 126 is Ala,and amino acid 16 is Gly. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ile, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Asn,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Ala,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Lys,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is Arg,and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesan amino acid sequence having at least 90%, at least 95%, at least 98%,or at least 99%, amino acid sequence identity to the amino acid sequencedepicted in FIG. 2Q, where amino acid 42 is Ala, amino acid 126 is His,and amino acid 16 is Ala. In some cases, a single copy of the variantIL-2 polypeptide is present in a multimeric polypeptide of the presentdisclosure. In some cases, a multimeric polypeptide of the presentdisclosure comprises two copies of the variant IL-2 polypeptide, e.g.,where the two copies are in tandem with no linker between the twocopies, or are in tandem and separated by a linker peptide. In somecases, a multimeric polypeptide of the present disclosure comprisesthree copies of the variant IL-2 polypeptide, e.g., where the threecopies are in tandem with no linker between the three copies, or are intandem and separated by a linker peptide. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand D2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the HLA Class I heavy chain. In some cases, where anIL-2/synTac of the present disclosure comprises HLA Class I heavy chainand D2M, the IL-2 polypeptide(s) is/are on the polypeptide chaincomprising the β2M polypeptide. In some cases, the variant IL-2polypeptide, or a synTac comprising the variant IL-2 polypeptide, has abinding affinity for IL-2R that is from about 100 nM to 150 nM, fromabout 150 nM to about 200 nM, from about 200 nM to about 250 nM, fromabout 250 nM to about 300 nM, from about 300 nM to about 350 nM, fromabout 350 nM to about 400 nM, from about 400 nM to about 500 nM, fromabout 500 nM to about 600 nM, from about 600 nM to about 700 nM, fromabout 700 nM to about 800 nM, from about 800 nM to about 900 nM, fromabout 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μMto about 10 μM, from about 10 μM to about 15 μM, from about 15 μM toabout 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100μM. In some cases, the variant IL-2 polypeptide has a length of 133amino acids.

Multiple Immunomodulatory Domains

As noted above, in some cases, a multimeric polypeptide of the presentdisclosure comprises two or more immunomodulatory polypeptides, where atleast one of the two or more immunomodulatory polypeptide is a variantIL-2 polypeptide of the present disclosure.

In some cases, a multimeric polypeptide of the present disclosurecomprises two or more copies of a variant IL-2 polypeptide of thepresent disclosure. In some cases, the two or more variant IL-2polypeptides are on the same polypeptide chain of a multimericpolypeptide of the present disclosure. In some cases, the two or morevariant IL-2 polypeptides are on separate polypeptide chains of amultimeric polypeptide of the present disclosure.

In some cases, a multimeric polypeptide of the present disclosurecomprises a first immunomodulatory polypeptide, and at least a secondimmunomodulatory polypeptide, where the first immunomodulatorypolypeptide is a variant IL-2 polypeptide of the present disclosure, andthe second immunomodulatory polypeptide is not an IL-2 polypeptide. Forexample, in some cases, the second immunomodulatory polypeptide is amember of the tumor necrosis factor (TNF) superfamily; e.g., a FasLpolypeptide, a 4-1BBL polypeptide, a CD40 polypeptide, an OX40Lpolypeptide, a CD30L polypeptide, a CD70 polypeptide, etc. In somecases, the second immunomodulatory polypeptide of a multimericpolypeptide of the present disclosure is a T-cell co-stimulatorypolypeptide and is a member of the immunoglobulin (Ig) superfamily;e.g., a CD7 polypeptide, a CD86 polypeptide, an ICAM polypeptide, etc.In some cases, the second immunomodulatory polypeptide is 4-1BBL, OX40L,ICOS-L, ICAM, PD-L1, CD86, FasL, and PD-L2. Suitable immunomodulatorypolypeptides of a multimeric polypeptide of the present disclosureinclude, e.g., CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, or HVEM. In some cases,the second immunomodulatory polypeptide is a variant (e.g., a variant ofnaturally-occurring 4-1BBL) that exhibits an affinity (determined asdescribed above) for its counterpart costimulatory protein found on theT cell that is reduced as compared to the affinity of the naturallyoccurring costimulatory protein (Immunomodulatory polypeptide) for itscounterpart (cognate) costimulatory protein. In some cases, a multimericpolypeptide of the present disclosure comprises a first immunomodulatorypolypeptide, and at least a second immunomodulatory polypeptide, whereinneither is a variant IL-2 polypeptide. It should be understood that thisdisclosure relates generally to the use of immunomodulatory polypeptidesthat are variants of naturally occurring immunomodulatory polypeptides,which variants exhibit an affinity (determined as described above) forcounterpart costimulatory proteins that is reduced as compared to theaffinity of the naturally occurring costimulatory protein(immunomodulatory polypeptide) for the counterpart (cognate)costimulatory protein.

Further T cell modulatory domains (MODs) that can be included in amultimeric polypeptide of the present disclosure include naturallyoccurring or synthetic human gene products (protein), affinity reagents(e.g., an antibody, antibody fragment, single chain Fvs, aptamers,nanobody) targeting a human gene product, including, but not limited toall secreted proteins arising from classical and non-classical (e.g.,FGF2, IL1, S100A4) secretion mechanisms, and ecto-domains of all cellsurface proteins anchored by naturally occurring genetically encodedprotein segments (single or multiple membrane spans) orpost-translational modifications such as GPI linkages). Any naturallyoccurring or synthetic affinity reagent (e.g., antibody, antibodyfragment, single chain Fvs, aptamer, nanobody, lectin, etc) targeting acell surface glycan or other post-translational modification (e.g.,sulfation). Examples include, but are not limited to, members of theTNF/TNFR family (OX40L, ICOSL, FASL, LTA, LTB TRAIL, CD153, TNFSF9,RANKL, TWEAK, TNFSF13, TNFSF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG,CD70) or affinity reagents directed at the TNF/INFR family members;members of the Immunoglobulin superfamily (VISTA, PD1, PD-L1, PD-L2,B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T cell receptor chains,ICOS, ICOS ligand, HHLA2, butyrophilins, BTLA, B7-H3, B7-H4, CD3, CD79a,CD79b, IgSF CAMS (including CD2, CD58, CD48, CD150, CD229, CD244,ICAM-1), Leukocyte immunoglobulin like receptors (LILR), killer cellinmunoglobulin like receptors (KIR)), lectin superfamily members,selectins, cytokines/chemokine and cytokine/chemokine receptors, growthfactors and growth factor receptors), adhesion molecules (integrins,fibronectins, cadherins), or ecto-domains of multi-span integralmembrane protein, or affinity reagents directed at the Immunoglobulinsuperfamily and listed gene products. In addition, activehomologs/orthologs of these gene products, including but not limited to,viral sequences (e.g., CMV, EBV), bacterial sequences, fungal sequences,eukaryotic pathogens (e.g., Schistosoma, Plasmodium, Babesia, Eimeria,Theileria, Toxoplasma, Entamoeba, Leishmania, and Trypanosonma), andmammalian-derived coding regions. In addition, a MOD may comprise asmall molecules drug targeting a human gene product.

Scaffold Polypeptides

A T-cell modulatory multimeric polypeptide of the present disclosurecomprises an Fc polypeptide, or another suitable scaffold polypeptide.

Suitable scaffold polypeptides include antibody-based scaffoldpolypeptides and non-antibody-based scaffolds. Non-antibody-basedscaffolds include, e.g., albumin, an XTEN (extended recombinant)polypeptide, transferrin, an Fc receptor polypeptide, an elastin-likepolypeptide (see, e.g., Hassouneh et al. (2012) Methods Enzymol.502:215; e.g., a polypeptide comprising a pentapeptide repeat unit of(Val-Pro-Gly-X-Gly; SEQ ID NO:100), where X is any amino acid other thanproline), an albumin-binding polypeptide, a silk-like polypeptide (see,e.g., Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci.357:165), a silk-elastin-like polypeptide (SELP; see, e.g., Megeed etal. (2002) Adv Drug Deliv Rev. 54:1075), and the like. Suitable XTENpolypeptides include, e.g., those disclosed in WO 2009/023270, WO2010/091122, WO 2007/103515, US 2010/0189682, and US 2009/0092582; seealso Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitablealbumin polypeptides include, e.g., human serum albumin.

Suitable scaffold polypeptides will in some cases be a half-lifeextending polypeptides. Thus, in some cases, a suitable scaffoldpolypeptide increases the in vivo half-life (e.g., the serum half-life)of the multimeric polypeptide, compared to a control multimericpolypeptide lacking the scaffold polypeptide. For example, in somecases, a scaffold polypeptide increases the in vivo half-life (e.g., theserum half-life) of the multimeric polypeptide, compared to a controlmultimeric polypeptide lacking the scaffold polypeptide, by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 50%, at least about 2-fold, at least about 2.5-fold, atleast about 5-fold, at least about 10-fold, at least about 25-fold, atleast about 50-fold, at least about 100-fold, or more than 100-fold. Asan example, in some cases, an Fc polypeptide increases the in vivohalf-life (e.g., the serum half-life) of the multimeric polypeptide,compared to a control multimeric polypeptide lacking the Fe polypeptide,by at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 50%, at least about 2-fold, at least about2.5-fold, at least about 5-fold, at least about 10-fold, at least about25-fold, at least about 50-fold, at least about 100-fold, or more than100-fold.

Fc Polypeptides

In some cases, the first and/or the second polypeptide chain of amultimeric polypeptide of the present disclosure comprises an Fcpolypeptide. The Fc polypeptide of a multimeric polypeptide of thepresent disclosure can be a human IgG1 Fe, a human IgG2 Fc, a human IgG3Fc, a human IgG4 Fc, etc. In some cases, the Fc polypeptide comprises anamino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to an amino acid sequence of an Fc region depicted inFIGS. 4A-C. In some cases, the Fc region comprises an amino acidsequence having at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the human IgG1 Fc polypeptide depicted in FIG. 4A. In somecases, the Fc region comprises an amino acid sequence having at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the human IgG1 Fcpolypeptide depicted in FIG. 4A; and comprises a substitution of N77;e.g., the Fc polypeptide comprises an N77A substitution. In some cases,the Fc polypeptide comprises an amino acid sequence having at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to the human IgG2 Fcpolypeptide depicted in FIG. 4A; e.g., the Fc polypeptide comprises anamino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 99-325 of the human IgG2 Fc polypeptidedepicted in FIG. 4A. In some cases, the Fc polypeptide comprises anamino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the human IgG3 Fc polypeptide depicted in FIG. 4A;e.g., the Fc polypeptide comprises an amino acid sequence having atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids19-246 of the human IgG3 Fc polypeptide depicted in FIG. 4A. In somecases, the Fc polypeptide comprises an amino acid sequence having atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to the human IμMFc polypeptide depicted in FIG. 4B; e.g., the Fe polypeptide comprisesan amino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 1-276 to the human IμM Fc polypeptidedepicted in FIG. 4B. In some cases, the Fc polypeptide comprises anamino acid sequence having at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the human IgA Fc polypeptide depicted in FIG. 4C;e.g., the Fc polypeptide comprises an amino acid sequence having atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids1-234 to the human IgA Fc polypeptide depicted in FIG. 4C.

In some cases, the Fc polypeptide present in a multimeric polypeptide ofthe present disclosure comprises the amino acid sequence depicted inFIG. 33A (human IgG1 Fc). In some cases, the Fc polypeptide present in amultimeric polypeptide of the present disclosure comprises the aminoacid sequence depicted in FIG. 33A (human IgG1 Fe), except for asubstitution of N297 with an amino acid other than asparagine. In somecases, the Fe polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence depicted in FIG.33C (human IgG1 Fc comprising an N297A substitution). In some cases, theFc polypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence depicted in FIG. 33A (humanIgG1 Fc), except for a substitution of L234 with an amino acid otherthan leucine. In some cases, the Fc polypeptide present in a multimericpolypeptide of the present disclosure comprises the amino acid sequencedepicted in FIG. 33A (human IgG1 Fc), except for a substitution of L235with an amino acid other than leucine. In some cases, the Fc polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence depicted in FIG. 33D (human IgG1 Fe comprisingan L234A substitution and an L235A substitution). In some cases, the Fcpolypeptide present in a multimeric polypeptide of the presentdisclosure comprises the amino acid sequence depicted in FIG. 33A (humanIgG1 Fc), except for a substitution of P331 with an amino acid otherthan proline; in some cases, the substitution is a P331S substitution.In some cases, the Fe polypeptide present in a multimeric polypeptide ofthe present disclosure comprises the amino acid sequence depicted inFIG. 33A (human IgG1 Fc), except for substitutions at L234 and L235 withamino acids other than leucine. In some cases, the Fc polypeptidepresent in a multimeric polypeptide of the present disclosure comprisesthe amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except forsubstitutions at L234 and L235 with amino acids other than leucine, anda substitution of P331 with an amino acid other than proline. In somecases, the Fc polypeptide present in a multimeric polypeptide of thepresent disclosure comprises the amino acid sequence depicted in FIG.33B (human IgG1 Fc comprising L234F, L235E, and P331S substitutions).

Additional Polypeptides

A polypeptide chain of a multimeric polypeptide of the presentdisclosure can include one or more polypeptides in addition to thosedescribed above. Suitable additional polypeptides include epitope tagsand affinity domains. The one or more additional polypeptide can beincluded at the N-terminus of a polypeptide chain of a multimericpolypeptide of the present disclosure, at the C-terminus of apolypeptide chain of a multimeric polypeptide of the present disclosure,or internally within a polypeptide chain of a multimeric polypeptide ofthe present disclosure.

Epitope Tag

Suitable epitope tags include, but are not limited to, hemagglutinin(HA; e.g., YPYDVPDYA (SEQ ID NO:20); FLAG (e.g., DYKDDDDK (SEQ IDNO:21); c-myc (e.g., EQKLISEEDL; SEQ ID NO:22), and the like.

Affinity Domain

Affinity domains include peptide sequences that can interact with abinding partner, e.g., such as one immobilized on a solid support,useful for identification or purification. DNA sequences encodingmultiple consecutive single amino acids, such as histidine, when fusedto the expressed protein, may be used for one-step purification of therecombinant protein by high affinity binding to a resin column, such asnickel sepharose. Exemplary affinity domains include His5 (HHHHH) (SEQID NO:23), HisX6 (HHHHHH) (SEQ ID NO:24), C-myc (EQKLISEEDL) (SEQ IDNO:22), Flag (DYKDDDDK) (SEQ ID NO:21), StrepTag (WSHPQFEK) (SEQ IDNO:25), hemagglutinin, e.g., HA Tag (YPYDVPDYA) (SEQ ID NO:20),glutathione-S-transferase (GST), thioredoxin, cellulose binding domain,RYIRS (SEQ ID NO:26), Phe-His-His-Thr (SEQ ID NO:88), chitin bindingdomain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag,WEAAAREACCRECCARA (SEQ ID NO:27), metal binding domains, e.g., zincbinding domains or calcium binding domains such as those fromcalcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B,myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin,hippocalcin, frequenin, caltractin, calpain large-subunit, S100proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin,inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, andmaltose binding protein.

Examples of Multimeric Polypeptides of the Present Disclosure

The following are non-limiting embodiments of an IL-2/synTac multimericpolypeptide of the present disclosure.

In some cases, an IL-2/synTac multimeric polypeptide of the presentdisclosure comprises: a) a first polypeptide comprising, in order fromN-terminus to C-terminus; i) an epitope; ii) a β2-microglobulin (β2M)polypeptide comprising the amino acid sequence depicted in FIG. 34A; andb) a second polypeptide comprising, in order from N-terminus toC-terminus; i) a variant IL-2 polypeptide of the present disclosure; ii)a major histocompatibility complex (MHC) heavy chain polypeptidecomprising the amino acid sequence depicted in FIG. 34C; and iii) anIgG1 Fc polypeptide comprising one or more amino acid substitutionsselected from N297A, L234A, L235A, L234F, L235E, and P331S. In somecases, the variant IL-2 polypeptide comprises an H16A and an F42Asubstitution. In some cases, the IgG1 Fc polypeptide comprises an N297Asubstitution. In some cases, the IgG1 Fc polypeptide comprises an L234Asubstitution and an L235A substitution. In some cases, the IgG1 Fcpolypeptide comprises an L234F substitution and an L235E substitution.In some cases, the IgG1 Fe polypeptide comprises an L234F substitution,an L235E substitution, and a P331S substitution. In some cases, thesecond polypeptide comprises two copies of the variant IL-2 polypeptide.In some cases, the first polypeptide comprises a peptide linker betweenthe epitope and the β2M polypeptide. In some cases, the secondpolypeptide comprises a peptide linker between one or more of: a) afirst copy of the variant IL-2 polypeptide and a second copy of thevariant IL-2 polypeptide; b) the variant IL-2 polypeptide and the MHCheavy chain polypeptide; and c) between the MHC heavy chain polypeptideand the IgG1 Fc polypeptide. In some cases, the peptide linker isselected from (GCGGG)₃ (SEQ ID NO:89), (GGGGS)₄ (SEQ ID NO:90), andAAACGG (SEQ ID NO:28). In some cases, the IgG1 Fc polypeptide comprisesthe amino acid sequence depicted in FIG. 33B. In some cases, the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33C. Insome cases, the IgG1 Fc polypeptide comprises the amino acid sequencedepicted in FIG. 33D.

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus; i) an epitope; ii) a β2-microglobulin polypeptidecomprising the amino acid sequence depicted in FIG. 34A; and b) a secondpolypeptide comprising, in order from N-terminus to C-terminus; i) avariant IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 34B; ii) a major histocompatibility complex (MHC) heavy chainpolypeptide comprising the amino acid sequence depicted in FIG. 34C; andiii) an IgG1 Fc polypeptide comprising one or more amino acidsubstitutions selected from N297A, L234A, L235A, L234F, L235E, andP331S. In some cases, the IgG1 Fc polypeptide comprises an N297Asubstitution. In some cases, the IgG1 Fe polypeptide comprises an L234Asubstitution and an L235A substitution. In some cases, the IgG1 Fcpolypeptide comprises an L234F substitution and an L235E substitution.In some cases, the IgG1 Fe polypeptide comprises an L234F substitution,an L235E substitution, and a P331S substitution. In some cases, the IgG1Fc polypeptide comprises the amino acid sequence depicted in FIG. 33B.In some cases, the IgG1 Fc polypeptide comprises the amino acid sequencedepicted in FIG. 33C. In some cases, the IgG1 Fc polypeptide comprisesthe amino acid sequence depicted in FIG. 33D. In some cases, in thesecond polypeptide comprises two copies of the variant IL-2 polypeptide.In some cases, the first polypeptide comprises a peptide linker betweenthe epitope and the β2M polypeptide. In some cases, the secondpolypeptide comprises a peptide linker between one or more of: a) afirst copy of the variant IL-2 polypeptide and a second copy of thevariant IL-2 polypeptide; b) the variant IL-2 polypeptide and the MHCheavy chain polypeptide; and c) between the MHC heavy chain polypeptideand the IgG1 Fc polypeptide. In some cases, the peptide linker isselected f nm (GGGGS)₃ (SEQ ID NO:89), (GGGGS)₄ (SEQ ID NO:90), andAAAGG (SEQ ID NO:28).

In some cases, multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising, in order from N-terminusto C-terminus; i) an epitope comprising the amino acid sequenceYMLDLQPETT (SEQ ID NO:13); ii) a β2-microglobulin polypeptide comprisingthe amino acid sequence depicted in FIG. 34A; and b) a secondpolypeptide comprising, in order from N-terminus to C-terminus; i) avariant IL-2 polypeptide comprising the amino acid sequence depicted inFIG. 34B; ii) a major histocompatibility complex (MHC) heavy chainpolypeptide comprising the amino acid sequence depicted in FIG. 34C; andiii) an IgG1 Fc polypeptide comprising the amino acid sequence depictedin FIG. 33A, 33B, 33C, or 33D. In some cases, the IgG1 Fc polypeptidecomprises the amino acid sequence depicted in FIG. 33B. In some cases,the IgG1 Fc polypeptide comprises the amino acid sequence depicted inFIG. 33C. In some cases, the IgG1 Fc polypeptide comprises the aminoacid sequence depicted in FIG. 33D. In some cases, the secondpolypeptide comprises two copies of the variant IL-2 polypeptide. Insome cases, the first polypeptide comprises a peptide linker between theepitope and the β2M polypeptide. In some cases, the second polypeptidecomprises a peptide linker between one or more of: a) a first copy ofthe variant IL-2 polypeptide and a second copy of the variant IL-2polypeptide; b) the variant IL-2 polypeptide and the MHC heavy chainpolypeptide; and c) between the MHC heavy chain polypeptide and the IgG1Fc polypeptide. In some cases, the peptide linker is selected from(GGGGS) (SEQ ID NO:89), (GGGGS)₄ (SEQ ID NO:90), and AAAGG (SEQ IDNO:28). In some cases, the IgG1 Fc polypeptide comprises the amino acidsequence depicted in FIG. 33B. In some cases, the IgG1 Fc polypeptidecomprises the amino acid sequence depicted in FIG. 33C. In some cases,the IgG1 Fc polypeptide comprises the amino acid sequence depicted inFIG. 33D.

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising the amino acid sequencedepicted in FIG. 31; and b) a second polypeptide comprising the aminoacid sequence depicted in FIG. 22.

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising the amino acid sequencedepicted in FIG. 31; and b) a second polypeptide comprising the aminoacid sequence depicted in FIG. 25.

In some cases, a multimeric polypeptide of the present disclosurecomprises: a) a first polypeptide comprising the amino acid sequencedepicted in FIG. 31; and ab) a second polypeptide comprising the aminoacid sequence depicted in FIG. 28.

Nucleic Acids

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a variant IL-2 polypeptide of the present disclosure.The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding an IL-2 fusion polypeptide of the present disclosure.

The present disclosure provides nucleic acids comprising nucleotidesequences encoding a multimeric polypeptide of the present disclosure.In some cases, the individual polypeptide chains of a multimericpolypeptide of the present disclosure are encoded in separate nucleicacids. In some cases, all polypeptide chains of a multimeric polypeptideof the present disclosure are encoded in a single nucleic acid. In somecases, a first nucleic acid comprises a nucleotide sequence encoding afirst polypeptide of a multimeric polypeptide of the present disclosure;and a second nucleic acid comprises a nucleotide sequence encoding asecond polypeptide of a multimeric polypeptide of the presentdisclosure. In some cases, single nucleic acid comprises a nucleotidesequence encoding a first polypeptide of a multimeric polypeptide of thepresent disclosure and a second polypeptide of a multimeric polypeptideof the present disclosure.

Non-limiting examples of nucleic acids of the present disclosure aredepicted in FIG. 23A. FIG. 26A, FIG. 29A, and FIG. 32.

Separate Nucleic Acids Encoding Individual Polypeptide Chains of aMultimeric Polypeptide

The present disclosure provides nucleic acids comprising nucleotidesequences encoding a multimeric polypeptide of the present disclosure.As noted above, in some cases, the individual polypeptide chains of amultimeric polypeptide of the present disclosure are encoded in separatenucleic acids. In some cases, nucleotide sequences encoding the separatepolypeptide chains of a multimeric polypeptide of the present disclosureare operably linked to transcriptional control elements, e.g.,promoters, such as promoters that are functional in a eukaryotic cell,where the promoter can be a constitutive promoter or an induciblepromoter.

The present disclosure provides a first nucleic acid and a secondnucleic acid, where the first nucleic acid comprises a nucleotidesequence encoding a first polypeptide of a multimeric polypeptide of thepresent disclosure, where the first polypeptide comprises, in order fromN-terminus to C-terminus; a) an epitope (e.g., a T-cell epitope); b) afirst MHC polypeptide; and c) an immunomodulatory polypeptide (e.g., avariant IL-2 polypeptide of the present disclosure); and where thesecond nucleic acid comprises a nucleotide sequence encoding a secondpolypeptide of a multimeric polypeptide of the present disclosure, wherethe second polypeptide comprises, in order from N-terminus toC-terminus; a) a second MHC polypeptide; and b) an Ig Fc polypeptide.Suitable T-cell epitopes, MHC polypeptides, immunomodulatorypolypeptides, and Ig Fe polypeptides, are described above. In somecases, the nucleotide sequences encoding the first and the secondpolypeptides are operably linked to transcriptional control elements. Insome cases, the transcriptional control element is a promoter that isfunctional in a eukaryotic cell. In some cases, the nucleic acids arepresent in separate expression vectors.

The present disclosure provides a first nucleic acid and a secondnucleic acid, where the first nucleic acid comprises a nucleotidesequence encoding a first polypeptide of a multimeric polypeptide of thepresent disclosure, where the first polypeptide comprises, in order fromN-terminus to C-terminus; a) an epitope (e.g., a T-cell epitope); and b)a first MHC polypeptide; and where the second nucleic acid comprises anucleotide sequence encoding a second polypeptide of a multimericpolypeptide of the present disclosure, where the second polypeptidecomprises, in order from N-terminus to C-terminus; a) animmunomodulatory polypeptide (e.g., a variant IL-2 polypeptide of thepresent disclosure); b) a second MHC polypeptide; and c) an Ig Fcpolypeptide. Suitable T-cell epitopes, MHC polypeptides,immunomodulatory polypeptides, and Ig Fe polypeptides, are describedabove. In some cases, the nucleotide sequences encoding the first andthe second polypeptides are operably linked to transcriptional controlelements. In some cases, the transcriptional control element is apromoter that is functional in a eukaryotic cell. In some cases, thenucleic acids are present in separate expression vectors.

Nucleic Acid Encoding Two or More Polypeptides Present in a MultimericPolypeptide

The present disclosure provides a nucleic acid comprising nucleotidesequences encoding at least the first polypeptide and the secondpolypeptide of a multimeric polypeptide of the present disclosure. Insome cases, where a multimeric polypeptide of the present disclosureincludes a first, second, and third polypeptide, the nucleic acidincludes a nucleotide sequence encoding the first, second, and thirdpolypeptides. In some cases, the nucleotide sequences encoding the firstpolypeptide and the second polypeptide of a multimeric polypeptide ofthe present disclosure includes a proteolytically cleavable linkerinterposed between the nucleotide sequence encoding the firstpolypeptide and the nucleotide sequence encoding the second polypeptide.In some cases, the nucleotide sequences encoding the first polypeptideand the second polypeptide of a multimeric polypeptide of the presentdisclosure includes an internal ribosome entry site (IRES) interposedbetween the nucleotide sequence encoding the first polypeptide and thenucleotide sequence encoding the second polypeptide. In some cases, thenucleotide sequences encoding the first polypeptide and the secondpolypeptide of a multimeric polypeptide of the present disclosureincludes a ribosome skipping signal (or cis-acting hydrolase element,CHYSEL) interposed between the nucleotide sequence encoding the firstpolypeptide and the nucleotide sequence encoding the second polypeptide.Examples of nucleic acids are described below, where a proteolyticallycleavable linker is provided between nucleotide sequences encoding thefirst polypeptide and the second polypeptide of a multimeric polypeptideof the present disclosure; in any of these embodiments, an IRES or aribosome skipping signal can be used in place of the nucleotide sequenceencoding the proteolytically cleavable linker.

In some cases, a first nucleic acid (e.g., a recombinant expressionvector, an mRNA, a viral RNA, etc.) comprises a nucleotide sequenceencoding a first polypeptide chain of a multimeric polypeptide of thepresent disclosure; and a second nucleic acid (e.g., a recombinantexpression vector, an mRNA, a viral RNA, etc.) comprises a nucleotidesequence encoding a second polypeptide chain of a multimeric polypeptideof the present disclosure. In some cases, the nucleotide sequenceencoding the first polypeptide, and the second nucleotide sequenceencoding the second polypeptide, are each operably linked totranscriptional control elements, e.g., promoters, such as promotersthat are functional in a eukaryotic cell, where the promoter can be aconstitutive promoter or an inducible promoter.

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a recombinant polypeptide, where the recombinantpolypeptide comprises, in order from N-terminus to C-terminus; a) anepitope (e.g., a T-cell epitope); b) a first MHC polypeptide; c) animmunomodulatory polypeptide (e.g., a variant IL-2 polypeptide of thepresent disclosure); d) a proteolytically cleavable linker; e) a secondMHC polypeptide; and f) an immunoglobulin (Ig) Fc polypeptide. Thepresent disclosure provides a nucleic acid comprising a nucleotidesequence encoding a recombinant polypeptide, where the recombinantpolypeptide comprises, in order from N-terminus to C-terminus; a) afirst leader peptide; b) the epitope; c) the first MHC polypeptide; d)the immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide ofthe present disclosure); e) the proteolytically cleavable linker; f) asecond leader peptide; g) the second MHC polypeptide; and h) the Ig Fcpolypeptide. The present disclosure provides a nucleic acid comprising anucleotide sequence encoding a recombinant polypeptide, where therecombinant polypeptide comprises, in order from N-terminus toC-terminus; a) an epitope; b) a first MHC polypeptide; c) aproteolytically cleavable linker; di an immunomodulatory polypeptide(e.g., a variant IL-2 polypeptide of the present disclosure); e) asecond MHC polypeptide; and f) an Ig Fe polypeptide. In some cases, thefirst leader peptide and the second leader peptide is a β2-M leaderpeptide. In some cases, the nucleotide sequence is operably linked to atranscriptional control element. In some cases, the transcriptionalcontrol element is a promoter that is functional in a eukaryotic cell.

Suitable MHC polypeptides are described above. In some cases, the firstMHC polypeptide is a β2-microglobulin polypeptide; and wherein thesecond MHC polypeptide is an MHC class I heavy chain polypeptide. Insome cases, the β2-microglobulin polypeptide comprises an amino acidsequence having at least 85% amino acid sequence identity to one of theamino acid sequences set forth in FIG. 6. In some cases, the MHC class Iheavy chain polypeptide is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G,HLA-K, or HLA-L heavy chain. In some cases, the MHC class I heavy chainpolypeptide comprises an amino acid sequence having at least 85% aminoacid sequence identity to the amino acid sequence set forth in one ofFIG. 5A-5C. In some cases, the first MHC polypeptide is an MHC Class IIalpha chain polypeptide; and wherein the second MHC polypeptide is anMHC class II beta chain polypeptide.

Suitable Fe polypeptides are described above. In some cases, the Ig Fcpolypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IμMFc polypeptide. In some cases, the Ig Fc polypeptide comprises an aminoacid sequence having at least 85% amino acid sequence identity to anamino acid sequence depicted in FIGS. 4A-4C.

Suitable Immunomodulatory Polypeptides are Described Above.

Suitable proteolytically cleavable linkers are described above. In somecases, the proteolytically cleavable linker comprises an amino acidsequence selected from: a) LEVLFQGP (SEQ ID NO:29); b) ENLYTQS (SEQ IDNO:30); c) DDDDK (SEQ ID NO:31); d) LVPR (SEQ ID NO:32); and e)GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:33).

In some cases, a linker between the epitope and the first MHCpolypeptide comprises a first Cys residue, and the second MHCpolypeptide comprises an amino acid substitution to provide a second Cysresidue, such that the first and the second Cys residues provide for adisulfide linkage between the linker and the second MHC polypeptide. Insome cases, first MHC polypeptide comprises an amino acid substitutionto provide a first Cys residue, and the second MHC polypeptide comprisesan amino acid substitution to provide a second Cys residue, such thatthe first Cys residue and the second Cys residue provide for a disulfidelinkage between the first MHC polypeptide and the second MHCpolypeptide.

Recombinant Expression Vectors

The present disclosure provides recombinant expression vectorscomprising nucleic acids of the present disclosure. In some cases, therecombinant expression vector is a non-viral vector. In someembodiments, the recombinant expression vector is a viral construct,e.g., a recombinant adeno-associated virus construct (see, e.g., U.S.Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinantlentiviral construct, a recombinant retroviral construct, anon-integrating viral vector, etc.

Suitable expression vectors include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549,1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999: WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomaiy et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like.

Numerous suitable expression vectors are known to those of skill in theart, and many are commercially available. The following vectors areprovided by way of example; for eukaryotic host cells; pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, anyother vector may be used so long as it is compatible with the host cell.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc, may be used in the expression vector(see e.g., Bitter et al. (1987) Methods in Enzymology. 153:516-544).

In some embodiments, a nucleotide sequence encoding a DNA-targeting RNAand/or a site-directed modifying polypeptide is operably linked to acontrol element, e.g., a transcriptional control element, such as apromoter. The transcriptional control element may be functional ineither a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell(e.g., bacterial or archaeal cell). In some embodiments, a nucleotidesequence encoding a DNA-targeting RNA and/or a site-directed modifyingpolypeptide is operably linked to multiple control elements that allowexpression of the nucleotide sequence encoding a DNA-targeting RNAand/or a site-directed modifying polypeptide in both prokaryotic andeukaryotic cells.

Non-limiting examples of suitable eukaryotic promoters (promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, andmouse metallothionein-1. Selection of the appropriate vector andpromoter is well within the level of ordinary skill in the art. Theexpression vector may also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector may also include appropriate sequences for amplifying expression.

Genetically Modified Host Cells

The present disclosure provides a genetically modified host cell, wherethe host cell is genetically modified with a nucleic acid of the presentdisclosure.

Suitable host cells include eukaryotic cells, such as yeast cells,insect cells, and mammalian cells. In some cases, the host cell is acell of a mammalian cell line. Suitable mammalian cell lines includehuman cell lines, non-human primate cell lines, rodent (e.g., mouse,rat) cell lines, and the like. Suitable mammalian cell lines include,but are not limited to, HeLa cells (e.g., American Type CultureCollection (ATCC) No. CCL-2)·CHO cells (e.g., ATCC Nos. CRL9618, CCL61,CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells(e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No.CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No.CRL1651), RAT1 cells, mouse L cells (ATCC No. CCL1.3), human embryonickidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

In some cases, the host cell is a mammalian cell that has beengenetically modified such that it does not synthesize endogenous MHCβ2-M.

Methods of Producing a Multimeric Polypeptide

The present disclosure provides methods of producing a multimericpolypeptide of the present disclosure. The methods generally involveculturing, in a culture medium, a host cell that is genetically modifiedwith a recombinant expression vector comprising a nucleotide sequenceencoding the multimeric polypeptide; and isolating the multimericpolypeptide from the genetically modified host cell and/or the culturemedium. A host cell that is genetically modified with a recombinantexpression vector comprising a nucleotide sequence encoding themultimeric polypeptide is also referred to as an “expression host.” Asnoted above, in some cases, the individual polypeptide chains of amultimeric polypeptide of the present disclosure are encoded in separaterecombinant expression vectors. In some cases, all polypeptide chains ofa multimeric polypeptide of the present disclosure are encoded in asingle recombinant expression vector.

Isolation of the multimeric polypeptide from the expression host cell(e.g., from a lysate of the expression host cell) and/or the culturemedium in which the host cell is cultured, can be carried out usingstandard methods of protein purification.

For example, a lysate may be prepared of the expression host and thelysate purified using high performance liquid chromatography (HPLC),exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique. Alternatively, where the multimericpolypeptide is secreted from the expression host cell into the culturemedium, the multimeric polypeptide can be purified from the culturemedium using HPLC, exclusion chromatography, gel electrophoresis,affinity chromatography, or other purification technique. In some cases,the compositions which are used will comprise at least 80% by weight ofthe desired product, at least about 85% by weight, at least about 95% byweight, or at least about 99.5% by weight, in relation to contaminantsrelated to the method of preparation of the product and itspurification. The percentages can be based upon total protein.

In some cases, e.g., where the multimeric polypeptide comprises anaffinity tag, the multimeric polypeptide can be purified using animmobilized binding partner of the affinity tag.

Compositions

The present disclosure provides compositions, including pharmaceuticalcompositions, comprising a variant IL-2 polypeptide of the presentdisclosure. The present disclosure provides compositions, includingpharmaceutical compositions, comprising a multimeric polypeptide of thepresent disclosure. The present disclosure provides compositions,including pharmaceutical compositions, comprising a nucleic acid or arecombinant expression vector of the present disclosure.

Compositions Comprising a Multimeric Polypeptide

A composition of the present disclosure can comprise, in addition to amultimeric polypeptide of the present disclosure, one or more of: asalt, e.g., NaCl, MgCl₂, KCl, MgSO₄, etc.; a buffering agent, e.g., aTris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)(HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; glycerol; and the like.

The composition may comprise a pharmaceutically acceptable excipient, avariety of which are known in the art and need not be discussed indetail herein. Pharmaceutically acceptable excipients have been amplydescribed in a variety of publications, including, for example,“Remington: The Science and Practice of Pharmacy”, 19^(th) Ed. (1995),or latest edition, Mack Publishing Co; A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy”, 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds 7^(th) ed., Lippincott, Williams, & Wilkins; andHandbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,3^(rd) ed. Amer. Pharmaceutical Assoc.

A pharmaceutical composition can comprise a multimeric polypeptide ofthe present disclosure, and a pharmaceutically acceptable excipient. Insome cases, a subject pharmaceutical composition will be suitable foradministration to a subject, e.g., will be sterile. For example, in someembodiments, a subject pharmaceutical composition will be suitable foradministration to a human subject, e.g., where the composition issterile and is free of detectable pyrogens and/or other toxins.

The protein compositions may comprise other components, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, hydrochloride,sulfate salts, solvates (e.g., mixed ionic salts, water, organics),hydrates (e.g., water), and the like.

For example, compositions may include aqueous solution, powder form,granules, tablets, pills, suppositories, capsules, suspensions, sprays,and the like. The composition may be formulated according to the variousroutes of administration described below.

Where a multimeric polypeptide of the present disclosure is administeredas an injectable (e.g. subcutaneously, intraperitoneally,intramuscularly, and/or intravenously) directly into a tissue, aformulation can be provided as a ready-to-use dosage form, or asnon-aqueous form (e.g., a reconstitutable storage-stable powder) oraqueous form, such as liquid composed of pharmaceutically acceptablecarriers and excipients. The protein-containing formulations may also beprovided so as to enhance serum half-life of the subject proteinfollowing administration. For example, the protein may be provided in aliposome formulation, prepared as a colloid, or other conventionaltechniques for extending serum half-life. A variety of methods areavailable for preparing liposomes, as described in, e.g., Szoka et al.1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871,4,501,728 and 4,837,028. The preparations may also be provided incontrolled release or slow-release forms.

Other examples of formulations suitable for parenteral administrationinclude isotonic sterile injection solutions, anti-oxidants,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. For example, asubject pharmaceutical composition can be present in a container, e.g.,a sterile container, such as a syringe. The formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets.

The concentration of a multimeric polypeptide of the present disclosurein a formulation can vary widely (e.g., from less than about 0.1%,usually at or at least about 2% to as much as 20% to 50% or more byweight) and will usually be selected primarily based on fluid volumes,viscosities, and patient-based factors in accordance with the particularmode of administration selected and the patient's needs.

The present disclosure provides a container comprising a composition ofthe present disclosure, e.g., a liquid composition. The container canbe, e.g., a syringe, an ampoule, and the like. In some cases, thecontainer is sterile. In some cases, both the container and thecomposition are sterile.

The present disclosure provides compositions, including pharmaceuticalcompositions, comprising a variant IL-2 polypeptide of the presentdisclosure. A composition can comprise: a) a variant IL-2 polypeptide ofthe present disclosure; and b) an excipient, as described above for themultimeric polypeptides. In some cases, the excipient is apharmaceutically acceptable excipient.

Compositions Comprising a Nucleic Acid or a Recombinant ExpressionVector

The present disclosure provides compositions, e.g., pharmaceuticalcompositions, comprising a nucleic acid or a recombinant expressionvector of the present disclosure. A wide variety of pharmaceuticallyacceptable excipients is known in the art and need not be discussed indetail herein. Pharmaceutically acceptable excipients have been amplydescribed in a variety of publications, including, for example, A.Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20thedition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Formsand Drug Delivery Systems (1999) H. C. Ansel et al., eds 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc.

A composition of the present disclosure can include: a) a subjectnucleic acid or recombinant expression vector; and b) one or more of: abuffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin,a chelating agent, a suspending agent, a solubilizer, a thickeningagent, a stabilizer, a bacteriostatic agent, a wetting agent, and apreservative. Suitable buffers include, but are not limited to, (such asN,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris),N-(2-hydroxyethyl)piperazine-N′3-propanesulfonic acid (EPPS or HEPPS),glycylglycine, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid(HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS),piperazine-N,N′-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate,3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid)TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-tris(hydroxymethyl)methyl-glycine (Tricine),tris(hydroxymethyl)-aminomethane (Tris), etc.). Suitable salts include,e.g., NaCl, MgCl₂, KCl, MgSO₄, etc.

A pharmaceutical formulation of the present disclosure can include anucleic acid or recombinant expression vector of the present disclosurein an amount of from about 0.001% to about 90% (w/w). In the descriptionof formulations, below, “subject nucleic acid or recombinant expressionvector” will be understood to include a nucleic acid or recombinantexpression vector of the present disclosure. For example, in someembodiments, a subject formulation comprises a nucleic acid orrecombinant expression vector of the present disclosure.

A subject nucleic acid or recombinant expression vector can be admixed,encapsulated, conjugated or otherwise associated with other compounds ormixtures of compounds; such compounds can include, e.g., liposomes orreceptor-targeted molecules. A subject nucleic acid or recombinantexpression vector can be combined in a formulation with one or morecomponents that assist in uptake, distribution and/or absorption.

A subject nucleic acid or recombinant expression vector composition canbe formulated into any of many possible dosage forms such as, but notlimited to, tablets, capsules, gel capsules, liquid syrups, soft gels,suppositories, and enemas. A subject nucleic acid or recombinantexpression vector composition can also be formulated as suspensions inaqueous, non-aqueous or mixed media. Aqueous suspensions may furthercontain substances which increase the viscosity of the suspensionincluding, for example, sodium carboxymethylcellulose, sorbitol and/ordextran. The suspension may also contain stabilizers.

A formulation comprising a subject nucleic acid or recombinantexpression vector can be a liposomal formulation. As used herein, theterm “liposome” means a vesicle composed of amphiphilic lipids arrangedin a spherical bilayer or bilayers. Liposomes are unilamellar ormultilamellar vesicles which have a membrane formed from a lipophilicmaterial and an aqueous interior that contains the composition to bedelivered. Cationic liposomes are positively charged liposomes that caninteract with negatively charged DNA molecules to form a stable complex.Liposomes that are pH sensitive or negatively charged are believed toentrap DNA rather than complex with it. Both cationic and noncationicliposomes can be used to deliver a subject nucleic acid or recombinantexpression vector.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein by reference in its entirety.

The formulations and compositions of the present disclosure may alsoinclude surfactants. The use of surfactants in drug products,formulations and in emulsions is well known in the art. Surfactants andtheir uses are further described in U.S. Pat. No. 6,287,860.

In one embodiment, various penetration enhancers are included, to effectthe efficient delivery of nucleic acids. In addition to aiding thediffusion of non-lipophilic drugs across cell membranes, penetrationenhancers also enhance the permeability of lipophilic drugs. Penetrationenhancers may be classified as belonging to one of five broadcategories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants. Penetration enhancers andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tablets,or minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Suitable oral formulationsinclude those in which a subject antisense nucleic acid is administeredin conjunction with one or more penetration enhancers surfactants andchelators. Suitable surfactants include, but are not limited to, fattyacids and/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts and fatty acids and their uses are furtherdescribed in U.S. Pat. No. 6,287,860. Also suitable are combinations ofpenetration enhancers, for example, fatty acids/salts in combinationwith bile acids/salts. An exemplary suitable combination is the sodiumsalt of lauric acid, capric acid, and UDCA. Further penetrationenhancers include, but are not limited to, polyoxyethylene-9-laurylether, and polyoxyethylene-20-cetyl ether. Suitable penetrationenhancers also include propylene glycol, dimethylsulfoxide,triethanoamine, N,N-dimethylacetamide, N,N-dimethylformamide,2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol, andAZONE™.

Methods of Modulating T Cell Activity

The present disclosure provides a method of selectively modulating theactivity of an epitope-specific T cell, the method comprising contactingthe T cell with a multimeric polypeptide of the present disclosure,where contacting the T cell with a multimeric polypeptide of the presentdisclosure selectively modulates the activity of the epitope-specific Tcell. In some cases, the contacting occurs in vitro. In some cases, thecontacting occurs in vivo. In some cases, the contacting occurs ex vivo.

In some cases, e.g., where the target T cell is a CD8⁺ T cell, themultimeric polypeptide comprises Class I MHC polypeptides (e.g.,β2-microglobulin and Class I MHC heavy chain). In some cases, e.g.,where the target T cell is a CD4⁺ T cell, the multimeric polypeptidecomprises Class II MHC polypeptides (e.g., Class II MHC α chain; ClassII MHC β chain).

Where a multimeric polypeptide of the present disclosure includes animmunomodulatory polypeptide that is an activating polypeptide,contacting the T cell with the multimeric polypeptide activates theepitope-specific T cell. In some instances, the epitope-specific T cellis a T cell that is specific for an epitope present on a cancer cell,and contacting the epitope-specific T cell with the multimericpolypeptide increases cytotoxic activity of the T cell toward the cancercell. In some instances, the epitope-specific T cell is a T cell that isspecific for an epitope present on a cancer cell, and contacting theepitope-specific T cell with the multimeric polypeptide increases thenumber of the epitope-specific T cells.

In some instances, the epitope-specific T cell is a T cell that isspecific for an epitope present on a virus-infected cell, and contactingthe epitope-specific T cell with the multimeric polypeptide increasescytotoxic activity of the T cell toward the virus-infected cell. In someinstances, the epitope-specific T cell is a T cell that is specific foran epitope present on a virus-infected cell, and contacting theepitope-specific T cell with the multimeric polypeptide increases thenumber of the epitope-specific T cells.

Where a multimeric polypeptide of the present disclosure includes animmunomodulatory polypeptide that is an inhibiting polypeptide,contacting the T cell with the multimeric inhibits the epitope-specificT cell. In some instances, the epitope-specific T cell is aself-reactive T cell that is specific for an epitope present in a selfantigen, and the contacting reduces the number of the self-reactive Tcells.

Methods of Selectively Delivering a Costimulatory Polypeptide (e.g.,IL-2)

The present disclosure provides a method of delivering a costimulatorypolypeptide such as IL-2, or a reduced-affinity variant of a naturallyoccurring costimulatory polypeptide such as an IL-2 variant disclosedherein, to a selected T cell or a selected T cell population, e.g., in amanner such that a TCR specific for a given epitope is targeted. Thepresent disclosure provides a method of delivering a costimulatorypolypeptide such as IL-2, or a reduced-affinity variant of a naturallyoccurring costimulatory polypeptide such as an IL-2 variant disclosedherein, selectively to a target T cell hearing a TCR specific for theepitope present in a multimeric polypeptide of the present disclosure.The method comprises contacting a population of T cells with amultimeric polypeptide of the present disclosure. The population of Tcells can be a mixed population that comprises: i) the target T cell;and ii) non-target T cells that are not specific for the epitope (e.g.,T cells that are specific for an epitope(s) other than the epitope towhich the epitope-specific T cell binds). The epitope-specific T cell isspecific for the epitope-presenting peptide present in the multimericpolypeptide, and binds to the peptide HLA complex or peptide MHC complexprovided by the multimeric polypeptide. Contacting the population of Tcells with the multimeric polypeptide delivers the costimulatorypolypeptide (e.g., IL-2 or a reduced-affinity variant of IL-2) presentin the multimeric polypeptide selectively to the T cell(s) that arespecific for the epitope present in the multimeric polypeptide.

Thus, the present disclosure provides a method of delivering acostimulatory polypeptide such as IL-2, or a reduced-affinity variant ofa naturally occurring costimulatory polypeptide such as an IL-2 variantdisclosed herein, or a combination of both, selectively to a target Tcell, the method comprising contacting a mixed population of T cellswith a multimeric polypeptide of the present disclosure. The mixedpopulation of T cells comprises the target T cell and non-target Tcells. The target T cell is specific for the epitope present within themultimeric polypeptide. Contacting the mixed population of T cells witha multimeric polypeptide of the present disclosure delivers thecostimulatory polypeptide(s) present within the multimeric polypeptideto the target T cell.

For example, a multimeric polypeptide of the present disclosure iscontacted with a population of T cells comprising: i) a target 1 cell(s)that is specific for the epitope present in the multimeric polypeptide;and ii) a non-target T cell(s), e.g., a T cell(s) that is specific for asecond epitope(s) that is not the epitope present in the multimericpolypeptide. Contacting the population results in selective delivery ofthe costimulatory polypeptide(s) (e.g., naturally-occurringcostimulatory polypeptide (e.g., naturally occurring IL-2) orreduced-affinity variant of a naturally occurring costimulatorypolypeptide (e.g., an IL-2 variant disclosed herein)), which is presentin the multimeric polypeptide, to the target T cell. Thus, e.g., lessthan 50%, less than 40%, less than 30%, less than 25%, less than 20%,less than 15%, less than 10%, less than 5%, or less than 4%, 3%, 2% or1%, of the non-target T cells bind the multimeric polypeptide and, as aresult, the costimulatory polypeptide (e.g., IL-2 or IL-2 variant) isnot delivered to the non-target T cells.

In some cases, the population of T cells is in vitro. In some cases, thepopulation of T cells is in vitro, and a biological response (e.g., Tcell activation and/or expansion and/or phenotypic differentiation) ofthe target T cell population to the multimeric polypeptide of thepresent disclosure is elicited in the context of an in vitro culture.For example, a mixed population of T cells can be obtained from anindividual, and can be contacted with the multimeric polypeptide invitro. Such contacting can comprise single or multiple exposures of thepopulation of T cells to a defined dose(s) and/or exposure schedule(s).In some cases, said contacting results in selectively binding/activatingand/or expanding target T cells within the population of T cells, andresults in generation of a population of activated and/or expandedtarget T cells. As an example, a mixed population of T cells can beperipheral blood mononuclear cells (PBMC). For example, PBMC from apatient can be obtained by standard blood drawing and PBMC enrichmenttechniques before being exposed to 0.1-1000 nM of a multimericpolypeptide of the present disclosure under standard lymphocyte cultureconditions. At time points before, during, and after exposure of themixed T cell population at a defined dose and schedule, the abundance oftarget T cells in the in vitro culture can be monitored by specificpeptide-MHC multimers and/or phenotypic markers and/or functionalactivity (e.g. cytokine ELISpot assays). In some cases, upon achievingan optimal abundance and/or phenotype of antigen specific cells invitro, all or a portion of the population of activated and/or expandedtarget T cells is administered to the individual (the individual fromwhom the mixed population of T cells was obtained).

In some cases, the population of T cells is in vitro. For example, amixed population of T cells is obtained from an individual, and iscontacted with a multimeric polypeptide of the present disclosure invitro. Such contacting, which can comprise single or multiple exposuresof the T cells to a defined dose(s) and/or exposure schedule(s) in thecontext of in vitro cell culture, can be used to determine whether themixed population of T cells includes T cells that are specific for theepitope presented by the multimeric polypeptide. The presence of T cellsthat are specific for the epitope of the multimeric polypeptide can bedetermined by assaying a sample comprising a mixed population of Tcells, which population of T cells comprises T cells that are notspecific for the epitope (non-target T cells) and may comprise T cellsthat are specific for the epitope (target T cells). Known assays can beused to detect activation and/or proliferation of the target T cells,thereby providing an ex vivo assay that can determine whether aparticular multimeric polypeptide (synTac) possesses an epitope thatbinds to T cells present in the individual and thus whether themultimeric polypeptide has potential use as a therapeutic compositionfor that individual. Suitable known assays for detection of activationand/or proliferation of target T cells include, e.g., flow cytometriccharacterization of T cell phenotype and/or antigen specificity and/orproliferation. Such an assay to detect the presence of epitope-specificT cells, e.g., a companion diagnostic, can further include additionalassays (e.g. effector cytokine ELISpot assays) and/or appropriatecontrols (e.g., antigen-specific and antigen-nonspecific multimericpeptide-HLA staining reagents) to determine whether the multimericpolypeptide is selectively binding/activating and/or expanding thetarget T cell. Thus, for example, the present disclosure provides amethod of detecting, in a mixed population of T cells obtained from anindividual, the presence of a target T cell that binds an epitope ofinterest, the method comprising: a) contacting in vitro the mixedpopulation of T cells with a multimeric polypeptide of the presentdisclosure, wherein the multimeric polypeptide comprises the epitope ofinterest; and b) detecting activation and/or proliferation of T cells inresponse to said contacting, wherein activated and/or proliferated Tcells indicates the presence of the target T cell. Alternatively, and/orin addition, if activation and/or expansion (proliferation) of thedesired T cell population is obtained using the multimeric polypeptide,then all or a portion of the population of T cells comprising theactivated/expanded T cells can be administered back to the individual asa therapy.

In some instances, the population of T cells is in vivo in anindividual. In such instances, a method of the present disclosure forselectively delivering a costimulatory polypeptide (e.g., IL-2 or areduced-affinity IL-2) to an epitope-specific T cell comprisesadministering the multimeric polypeptide to the individual.

The epitope-specific T cell to which a costimulatory polypeptide (e.g.,IL-2 or a reduced-affinity IL-2) is being selectively delivered is alsoreferred to herein as a “target T cell.” In some cases, the target Tcell is a regulatory T cell (Treg). In some cases, the Treg inhibits orsuppresses activity of an autoreactive T cell.

In some cases, the target T cell is a cytotoxic T cell. For example, thetarget T cell can be a cytotoxic T cell specific for a cancer epitope(e.g., an epitope presented by a cancer cell).

Treatment Methods

The present disclosure provides a method of selectively modulating theactivity of an epitope-specific T cell in an individual, the methodcomprising administering to the individual an amount of the multimericpolypeptide of the present disclosure, or one or more nucleic acidsencoding the multimeric polypeptide, effective to selectively modulatethe activity of an epitope-specific T cell in an individual. In somecases, a treatment method of the present disclosure comprisesadministering to an individual in need thereof one or more recombinantexpression vectors comprising nucleotide sequences encoding a multimericpolypeptide of the present disclosure. In some cases, a treatment methodof the present disclosure comprises administering to an individual inneed thereof one or more mRNA molecules comprising nucleotide sequencesencoding a multimeric polypeptide of the present disclosure. In somecases, a treatment method of the present disclosure comprisesadministering to an individual in need thereof a multimeric polypeptideof the present disclosure.

The present disclosure provides a method of selectively modulating theactivity of an epitope-specific T cell in an individual, the methodcomprising administering to the individual an effective amount of amultimeric polypeptide of the present disclosure, or one or more nucleicacids (e.g., expression vectors; mRNA; etc.) comprising nucleotidesequences encoding the multimeric polypeptide, where the multimericpolypeptide selectively modulates the activity of the epitope-specific Tcell in the individual. Selectively modulating the activity of anepitope-specific T cell can treat a disease or disorder in theindividual. Thus, the present disclosure provides a treatment methodcomprising administering to an individual in need thereof an effectiveamount of a multimeric polypeptide of the present disclosure.

In some cases, the immunomodulatory polypeptide is an activatingpolypeptide, and the multimeric polypeptide activates theepitope-specific T cell. In some cases, the epitope is acancer-associated epitope, and the multimeric polypeptide increases theactivity of a T cell specific for the cancer-associate epitope.

The present disclosure provides a method of treating cancer in anindividual, the method comprising administering to the individual aneffective amount of a multimeric polypeptide of the present disclosure,or one or more nucleic acids (e.g., expression vectors; mRNA; etc.)comprising nucleotide sequences encoding the multimeric polypeptide,where the multimeric polypeptide comprises a T-cell epitope that is acancer epitope, and where the multimeric polypeptide comprises one ormore stimulatory immunomodulatory polypeptides, as described herein. Insome cases, an “effective amount” of a multimeric polypeptide is anamount that, when administered in one or more doses to an individual inneed thereof, reduces the number of cancer cells in the individual. Forexample, in some cases, an “effective amount” of a multimericpolypeptide of the present disclosure is an amount that, whenadministered in one or more doses to an individual in need thereof,reduces the number of cancer cells in the individual by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95%, compared to the number of cancer cells in the individualbefore administration of the multimeric polypeptide, or in the absenceof administration with the multimeric polypeptide. In some cases, an“effective amount” of a multimeric polypeptide of the present disclosureis an amount that, when administered in one or more doses to anindividual in need thereof, reduces the number of cancer cells in theindividual to undetectable levels. In some cases, an “effective amount”of a multimeric polypeptide of the present disclosure is an amount that,when administered in one or more doses to an individual in need thereof,reduces the tumor mass in the individual. For example, in some cases, an“effective amount” of a multimeric polypeptide of the present disclosureis an amount that, when administered in one or more doses to anindividual in need thereof, reduces the tumor mass in the individual byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95%, compared to the tumor mass in the individualbefore administration of the multimeric polypeptide, or in the absenceof administration with the multimeric polypeptide. In some cases, an“effective amount” of a multimeric polypeptide of the present disclosureis an amount that, when administered in one or more doses to anindividual in need thereof, increases survival time of the individual.For example, in some cases, an “effective amount” of a multimericpolypeptide of the present disclosure is an amount that, whenadministered in one or more doses to an individual in need thereof,increases survival time of the individual by at least 1 month, at least2 months, at least 3 months, from 3 months to 6 months, from 6 months to1 year, from 1 year to 2 years, from 2 years to 5 years, from 5 years to10 years, or more than 10 years, compared to the expected survival timeof the individual in the absence of administration with the multimericpolypeptide.

In some instances, the epitope-specific T cell is a T cell that isspecific for an epitope present on a virus-infected cell, and contactingthe epitope-specific T cell with the multimeric polypeptide increasescytotoxic activity of the T cell toward the virus-infected cell. In someinstances, the epitope-specific T cell is a T cell that is specific foran epitope present on a virus-infected cell, and contacting theepitope-specific T cell with the multimeric polypeptide increases thenumber of the epitope-specific T cells.

Thus, the present disclosure provides a method of treating a virusinfection in an individual, the method comprising administering to theindividual an effective amount of a multimeric polypeptide of thepresent disclosure, or one or more nucleic acids comprising nucleotidesequences encoding the multimeric polypeptide, where the multimericpolypeptide comprises a T-cell epitope that is a viral epitope, andwhere the multimeric polypeptide comprises one or more stimulatoryimmunomodulatory polypeptides as described herein. In some cases, an“effective amount” of a multimeric polypeptide is an amount that, whenadministered in one or more doses to an individual in need thereof,reduces the number of virus-infected cells in the individual. Forexample, in some cases, an “effective amount” of a multimericpolypeptide of the present disclosure is an amount that, whenadministered in one or more doses to an individual in need thereof,reduces the number of virus-infected cells in the individual by at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or at least 95%, compared to the number of virus-infected cells inthe individual before administration of the multimeric polypeptide, orin the absence of administration with the multimeric polypeptide. Insome cases, an “effective amount” of a multimeric polypeptide of thepresent disclosure is an amount that, when administered in one or moredoses to an individual in need thereof, reduces the number ofvirus-infected cells in the individual to undetectable levels.

Thus, the present disclosure provides a method of treating an infectionin an individual, the method comprising administering to the individualan effective amount of a multimeric polypeptide of the presentdisclosure, or one or more nucleic acids comprising nucleotide sequencesencoding the multimeric polypeptide, where the multimeric polypeptidecomprises a T-cell epitope that is a pathogen-associated epitope, andwhere the multimeric polypeptide comprises one or more stimulatoryimmunomodulatory polypeptides as described herein. In some cases, an“effective amount” of a multimeric polypeptide is an amount that, whenadministered in one or more doses to an individual in need thereof,reduces the number of pathogens in the individual. For example, in somecases, an “effective amount” of a multimeric polypeptide of the presentdisclosure is an amount that, when administered in one or more doses toan individual in need thereof, reduces the number of pathogens in theindividual by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%, compared to the number ofpathogens in the individual before administration of the multimericpolypeptide, or in the absence of administration with the multimericpolypeptide. In some cases, an “effective amount” of a multimericpolypeptide of the present disclosure is an amount that, whenadministered in one or more doses to an individual in need thereof,reduces the number of pathogens in the individual to undetectablelevels. Pathogens include viruses, bacteria, protozoans, and the like.

In some cases, the immunomodulatory polypeptide is an inhibitorypolypeptide, and the multimeric polypeptide inhibits activity of theepitope-specific T cell. In some cases, the epitope is a self-epitope,and the multimeric polypeptide selectively inhibits the activity of a Tcell specific for the self-epitope.

The present disclosure provides a method of treating an autoimmunedisorder in an individual, the method comprising administering to theindividual an effective amount of a multimeric polypeptide of thepresent disclosure, or one or more nucleic acids comprising nucleotidesequences encoding the multimeric polypeptide, where the multimericpolypeptide comprises a T-cell epitope that is a self epitope, and wherethe multimeric polypeptide comprises an inhibitory immunomodulatorypolypeptide. In some cases, an “effective amount” of a multimericpolypeptide is an amount that, when administered in one or more doses toan individual in need thereof, reduces the number self-reactive T cellsby at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95%, compared to number of self-reactive T cellsin the individual before administration of the multimeric polypeptide,or in the absence of administration with the multimeric polypeptide. Insome cases, an “effective amount” of a multimeric polypeptide is anamount that, when administered in one or more doses to an individual inneed thereof, reduces production of Th2 cytokines in the individual. Insome cases, an “effective amount” of a multimeric polypeptide is anamount that, when administered in one or more doses to an individual inneed thereof, ameliorates one or more symptoms associated with anautoimmune disease in the individual.

As noted above, in some cases, in carrying out a subject treatmentmethod, a multimeric polypeptide of the present disclosure isadministered to an individual in need thereof, as the polypeptide perse. In other instances, in carrying out a subject treatment method, oneor more nucleic acids comprising nucleotide sequences encoding amultimeric polypeptide of the present disclosure is/are administering toan individual in need thereof. Thus, in other instances, one or morenucleic acids of the present disclosure, e.g., one or more recombinantexpression vectors of the present disclosure, is/are administered to anindividual in need thereof.

Formulations

Suitable formulations are described above, where suitable formulationsinclude a pharmaceutically acceptable excipient. In some cases, asuitable formulation comprises: a) a multimeric polypeptide of thepresent disclosure; and b) a pharmaceutically acceptable excipient. Insome cases, a suitable formulation comprises: a) a nucleic acidcomprising a nucleotide sequence encoding a multimeric polypeptide ofthe present disclosure; and b) a pharmaceutically acceptable excipient;in some instances, the nucleic acid is an mRNA. In some cases, asuitable formulation comprises: a) a first nucleic acid comprising anucleotide sequence encoding the first polypeptide of a multimericpolypeptide of the present disclosure; b) a second nucleic acidcomprising a nucleotide sequence encoding the second polypeptide of amultimeric polypeptide of the present disclosure; and c) apharmaceutically acceptable excipient. In some cases, a suitableformulation comprises: a) a recombinant expression vector comprising anucleotide sequence encoding a multimeric polypeptide of the presentdisclosure; and b) a pharmaceutically acceptable excipient. In somecases, a suitable formulation comprises: a) a first recombinantexpression vector comprising a nucleotide sequence encoding the firstpolypeptide of a multimeric polypeptide of the present disclosure; b) asecond recombinant expression vector comprising a nucleotide sequenceencoding the second polypeptide of a multimeric polypeptide of thepresent disclosure; and c) a pharmaceutically acceptable excipient.

Suitable pharmaceutically acceptable excipients are described above.

Dosages

A suitable dosage can be determined by an attending physician or otherqualified medical personnel, based on various clinical factors. As iswell known in the medical arts, dosages for any one patient depend uponmany factors, including the patient's size, body surface area, age, theparticular polypeptide or nucleic acid to be administered, sex of thepatient, time, and route of administration, general health, and otherdrugs being administered concurrently. A multimeric polypeptide of thepresent disclosure may be administered in amounts between 1 ng/kg bodyweight and 20 mg/kg body weight per dose, e.g. between 0.1 mg/kg bodyweight to 10 mg/kg body weight, e.g. between 0.5 mg/kg body weight to 5mg/kg body weight, however, doses below or above this exemplary rangeare envisioned, especially considering the aforementioned factors. Ifthe regimen is a continuous infusion, it can also be in the range of 1μg to 10 mg per kilogram of body weight per minute. A multimericpolypeptide of the present disclosure can be administered in an amountof from about 1 mg/kg body weight to 50 mg/kg body weight, e.g., fromabout 1 mg/kg body weight to about 5 mg/kg body weight, from about 5mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kgbody weight to about 15 mg/kg body weight, from about 15 mg/kg bodyweight to about 20 mg/kg body weight, from about 20 mg/kg body weight toabout 25 mg/kg body weight, from about 25 mg/kg body weight to about 30mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kgbody weight, from about 35 mg/kg body weight to about 40 mg/kg bodyweight, or from about 40 mg/kg body weight to about 50 mg/kg bodyweight.

In some cases, a suitable dose of a multimeric polypeptide of thepresent disclosure is from 0.01 μg to 100 g per kg of body weight, from0.1 μg to 10 g per kg of body weight, from 1 μg to 1 g per kg of bodyweight, from 10 μg to 100 mg per kg of body weight, from 100 μg to 10 mgper kg of body weight, or from 100 μg to 1 mg per kg of body weight.Persons of ordinary skill in the art can easily estimate repetitionrates for dosing based on measured residence times and concentrations ofthe administered agent in bodily fluids or tissues. Following successfultreatment, it may be desirable to have the patient undergo maintenancetherapy to prevent the recurrence of the disease state, wherein amultimeric polypeptide of the present disclosure is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight,from 0.1 μg to 10 g per kg of body weight, from 1 μg to 1 g per kg ofbody weight, from 10 μg to 100 mg per kg of body weight, from 100 μg to10 mg per kg of body weight, or from 100 μg to 1 mg per kg of bodyweight.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific multimeric polypeptide, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

In some embodiments, multiple doses of a multimeric polypeptide of thepresent disclosure, a nucleic acid of the present disclosure, or arecombinant expression vector of the present disclosure areadministered. The frequency of administration of a multimericpolypeptide of the present disclosure, a nucleic acid of the presentdisclosure, or a recombinant expression vector of the present disclosurecan vary depending on any of a variety of factors, e.g., severity of thesymptoms, etc. For example, in some embodiments, a multimericpolypeptide of the present disclosure, a nucleic acid of the presentdisclosure, or a recombinant expression vector of the presentdisclosure, is administered once per month, twice per month, three timesper month, every other week (qow), once per week (qw), twice per week(biw), three times per week (tiw), four times per week, five times perweek, six times per week, every other day (qod), daily (qd), twice a day(qid), or three times a day (tid).

The duration of administration of a multimeric polypeptide of thepresent disclosure, a nucleic acid of the present disclosure, or arecombinant expression vector of the present disclosure, e.g., theperiod of time over which a multimeric polypeptide of the presentdisclosure, a nucleic acid of the present disclosure, or a recombinantexpression vector of the present disclosure is administered, can vary,depending on any of a variety of factors, e.g., patient response, etc.For example, a multimeric polypeptide of the present disclosure, anucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure can be administered over a period oftime ranging from about one day to about one week, from about two weeksto about four weeks, from about one month to about two months, fromabout two months to about four months, from about four months to aboutsix months, from about six months to about eight months, from abouteight months to about 1 year, from about 1 year to about 2 years, orfrom about 2 years to about 4 years, or more.

Routes of Administration

An active agent (a multimeric polypeptide of the present disclosure, anucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure) is administered to an individual usingany available method and route suitable for drug delivery, including invivo and ex vivo methods, as well as systemic and localized routes ofadministration.

Conventional and pharmaceutically acceptable routes of administrationinclude intratumoral, peritumoral, intramuscular, intratracheal,intracranial, subcutaneous, intradermal, topical application,intravenous, intraarterial, rectal, nasal, oral, and other enteral andparenteral routes of administration. Routes of administration may becombined, if desired, or adjusted depending upon the multimericpolypeptide and/or the desired effect. A multimeric polypeptide of thepresent disclosure, or a nucleic acid or recombinant expression vectorof the present disclosure, can be administered in a single dose or inmultiple doses.

In some embodiments, a multimeric polypeptide of the present disclosure,a nucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure is administered intravenously. In someembodiments, a multimeric polypeptide of the present disclosure, anucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure is administered intramuscularly. Insome embodiments, a multimeric polypeptide of the present disclosure, anucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure is administered locally. In someembodiments, a multimeric polypeptide of the present disclosure, anucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure is administered intratumorally. In someembodiments, a multimeric polypeptide of the present disclosure, anucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure is administered peritumorally. In someembodiments, a multimeric polypeptide of the present disclosure, anucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure is administered intracranially. In someembodiments, a multimeric polypeptide of the present disclosure, anucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure is administered subcutaneously.

In some embodiments, a multimeric polypeptide of the present disclosureis administered intravenously. In some embodiments, a multimericpolypeptide of the present disclosure is administered intramuscularly.In some embodiments, a multimeric polypeptide of the present disclosureis administered locally. In some embodiments, a multimeric polypeptideof the present disclosure is administered intratumorally. In someembodiments, a multimeric polypeptide of the present disclosure isadministered peritumorally. In some embodiments, a multimericpolypeptide of the present disclosure is administered intracranially. Insome embodiments, a multimeric polypeptide is administeredsubcutaneously.

A multimeric polypeptide of the present disclosure, a nucleic acid ofthe present disclosure, or a recombinant expression vector of thepresent disclosure can be administered to a host using any availableconventional methods and routes suitable for delivery of conventionaldrugs, including systemic or localized routes. In general, routes ofadministration contemplated for use in a method of the presentdisclosure include, but are not necessarily limited to, enteral,parenteral, and inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, intratumoral, peritumoral, and intravenous mutes, i.e.,any route of administration other than through the alimentary canal.Parenteral administration can be carried to effect systemic or localdelivery of a multimeric polypeptide of the present disclosure, anucleic acid of the present disclosure, or a recombinant expressionvector of the present disclosure. Where systemic delivery is desired,administration typically involves invasive or systemically absorbedtopical or mucosal administration of pharmaceutical preparations.

Subjects Suitable for Treatment

Subjects suitable for treatment with a method of the present disclosureinclude individuals who have cancer, including individuals who have beendiagnosed as having cancer, individuals who have been treated for cancerbut who failed to respond to the treatment, and individuals who havebeen treated for cancer and who initially responded but subsequentlybecame refractory to the treatment. Subjects suitable for treatment witha method of the present disclosure include individuals who have aninfection (e.g., an infection with a pathogen such as a bacterium, avirus, a protozoan, etc.), including individuals who have been diagnosedas having an infection, and individuals who have been treated for aninfection but who failed to respond to the treatment. Subjects suitablefor treatment with a method of the present disclosure includeindividuals who have bacterial infection, including individuals who havebeen diagnosed as having a bacterial infection, and individuals who havebeen treated for a bacterial infection but who failed to respond to thetreatment. Subjects suitable for treatment with a method of the presentdisclosure include individuals who have a viral infection, includingindividuals who have been diagnosed as having a viral infection, andindividuals who have been treated for a viral infection but who failedto respond to the treatment. Subjects suitable for treatment with amethod of the present disclosure include individuals who have anautoimmune disease, including individuals who have been diagnosed ashaving an autoimmune disease, and individuals who have been treated fora autoimmune disease but who failed to respond to the treatment.

In some cases, e.g., where the epitope is an HPV epitope, a subjectsuitable for treatment with a method of the present disclosure is anindividual who has been diagnosed as having an HPV-associated cancer oran HPV-attributable cancer. HPV-associated and HPV-attributable cancersinclude, e.g., head and neck cancer; cervical cancer; and genitoanalcancer.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-132 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below:

Aspect 1. A variant IL-2 polypeptide comprising an amino acid sequencehaving at least 85% amino acid sequence identity to set forth in SEQ IDNO:1, wherein the variant IL-2 polypeptide has one or more amino acidsubstitutions relative to set forth in SEQ ID NO:1, and wherein thevariant IL-2 polypeptide exhibits reduced binding affinity to an IL-2receptor (IL-2R) comprising alpha, beta, and gamma polypeptides havingamino acid sequences depicted in FIG. 3A-3C, compared to the bindingaffinity of the IL-2 amino acid sequence set forth in one of SEQ ID NO:1for the IL-2R.

Aspect 2. The variant IL-2 polypeptide of aspect 1, wherein the variantcomprises a substitution of one or more of E15, H16, D20, F42, Y45, andQ126.

Aspect 3. The variant IL-2 polypeptide of aspect 1 or aspect 2, whereinthe variant immunomodulatory polypeptide exhibits from less than 10% toless than 50% of the binding affinity exhibited by the IL-2 amino acidsequence set forth in SEQ ID NO:1 for the IL-2R.

Aspect 4. The variant IL-2 polypeptide of any one of aspects 1-3,wherein the variant comprises substitutions of F42 with Ala, Gly, Val,Ile, or Leu.

Aspect 5. The variant IL-2 polypeptide of any one of aspects 1-3,wherein the variant comprises substitutions of F42 and D20 orsubstitutions of F42 and H16.

Aspect 6. The variant IL-2 polypeptide of any one of aspects 1-3,wherein the variant comprises substitutions of F42, D20, and Y45; orwherein the variant comprises substitutions of F42, H16, and Q126.

Aspect 7. A multimeric polypeptide comprising:

a) a first polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) an epitope;    -   ii) a first major histocompatibility complex (MHC) polypeptide;        and

b) a second polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) a second MHC polypeptide; and    -   ii) optionally an immunoglobulin (Ig) Fc polypeptide or a non-Ig        scaffold,

wherein the multimeric polypeptide comprises one or moreimmunomodulatory domains, wherein the one or more immunomodulatorydomain is:

-   -   A) at the C-terminus of the first polypeptide;    -   B) at the N-terminus of the second polypeptide;    -   C) at the C-terminus of the second polypeptide; or    -   D) at the C-terminus of the first polypeptide and at the        N-terminus of the second polypeptide, and

wherein at least one of the immunomodulatory domains is a variant of anaturally occurring costimulatory protein, and wherein the variantexhibits a reduced affinity for its counterpart costimulatory protein ascompared to the affinity of the naturally occurring costimulatoryprotein for the counterpart costimulatory protein.

Aspect 8. A multimeric polypeptide comprising:

a) a first polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) an epitope;    -   ii) a first major histocompatibility complex (MHC) polypeptide;        and

b) a second polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) a second MHC polypeptide; and    -   ii) optionally an immunoglobulin (Ig) Fc polypeptide or a non-Ig        scaffold,

wherein the multimeric polypeptide comprises one or moreimmunomodulatory domains, wherein the one or more immunomodulatorydomain is:

-   -   A) at the C-terminus of the first polypeptide;    -   B) at the N-terminus of the second polypeptide;    -   C) at the C-terminus of the second polypeptide; or    -   D) at the C-terminus of the first polypeptide and at the        N-terminus of the second polypeptide,

wherein at least one of the one or more immunomodulatory domains is avariant IL-2 polypeptide of any one of aspects 1-6, and

wherein the multimeric polypeptide exhibits reduced binding affinity toan IL-2 receptor (IL-2R) comprising alpha, beta, and gamma polypeptideshaving amino acid sequences depicted in FIG. 3A-3C, compared to thebinding affinity of a control multimeric polypeptide comprising the IL-2amino acid sequence set forth in SEQ ID NO:1 for the IL-2R polypeptide.

Aspect 9. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide comprises, in order from N-terminus toC-terminus;

-   -   i) the epitope;    -   ii) the first MHC polypeptide; and    -   iii) the variant IL-2 polypeptide; and

b) the second polypeptide comprises, in order from N-terminus toC-terminus;

-   -   i) the second MHC polypeptide; and    -   ii) the Ig Fc polypeptide.

Aspect 10. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide comprises, in order from N-terminus toC-terminus;

-   -   i) the epitope; and    -   ii) the first MHC polypeptide; and

b) the second polypeptide comprises, in order from N-terminus toC-terminus;

-   -   i) the variant IL-2 polypeptide;    -   ii) the second MHC polypeptide; and    -   iii) the Ig Fe polypeptide.

Aspect 11. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide comprises, in order from N-terminus toC-terminus;

-   -   i) the epitope; and    -   ii) the first MHC polypeptide; and

b) the second polypeptide comprises, in order from N-terminus toC-terminus;

-   -   i) the second MHC polypeptide; and    -   ii) the variant IL-2 polypeptide.

Aspect 12. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide comprises, in order from N-terminus toC-terminus;

-   -   i) the epitope; and    -   ii) the first MHC polypeptide; and

b) second polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) the variant IL-2 polypeptide; and    -   ii) the second MHC polypeptide.

Aspect 13. The multimeric polypeptide of aspect 8, wherein:

a) the first polypeptide comprises, in order from N-terminus toC-terminus;

-   -   i) the epitope;    -   ii) the first MHC polypeptide; and    -   iii) the variant IL-2 polypeptide; and

b) the second polypeptide comprises the second MHC polypeptide.

Aspect 14. The multimeric polypeptide of aspect 7 or 8, wherein thenon-Ig scaffold is an XTEN polypeptide, a transferrin polypeptide, anelastin-like polypeptide, a silk-like polypeptide, or asilk-elastin-like polypeptide.

Aspect 15. The multimeric polypeptide of any one of aspects 7-14,wherein the first MHC polypeptide is a β2-microglobulin polypeptide; andwherein the second MHC polypeptide is an MHC class I heavy chainpolypeptide.

Aspect 16. The multimeric polypeptide of aspect 15, wherein theβ2-microglobulin polypeptide comprises an amino acid sequence having atleast 85% amino acid sequence identity to one of the amino acidsequences set forth in FIG. 6.

Aspect 17. The multimeric polypeptide of aspect 15, wherein the MHCclass I heavy chain polypeptide is an HLA-A, an HLA-B, or an HLA-C heavychain.

Aspect 18. The multimeric polypeptide of aspect 15, wherein the MHCclass I heavy chain polypeptide comprises an amino acid sequence havingat least 85% amino acid sequence identity to the amino acid sequence setforth in one of FIG. 5A-5C.

Aspect 19. The multimeric polypeptide of any one of aspects 7-14,wherein the first MHC polypeptide is an MHC Class II alpha chainpolypeptide; and wherein the second MHC polypeptide is an MHC class IIbeta chain polypeptide.

Aspect 20. The multimeric polypeptide of any one of aspects 7-19,wherein the epitope is a T-cell epitope.

Aspect 21. The multimeric polypeptide of any one of aspects 7-13 and15-20, wherein multimeric polypeptide comprises an Fc polypeptide, andwherein the Ig Fe polypeptide is an IgG1 Fc polypeptide, an IgG2 Fcpolypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fcpolypeptide, or an IμM Fc polypeptide.

Aspect 22. The multimeric polypeptide of aspect 21, wherein the Ig Fcpolypeptide comprises an amino acid sequence having at least 85% aminoacid sequence identity to an amino acid sequence depicted in FIG. 4A-4C.

Aspect 23. The multimeric polypeptide of any one of aspects 7-22,wherein the first polypeptide and the second polypeptide arenon-covalently associated.

Aspect 24. The multimeric polypeptide of any one of aspects 7-22,wherein the first polypeptide and the second polypeptide are covalentlylinked to one another

Aspect 25. The multimeric polypeptide of aspect 24, wherein the covalentlinkage is via a disulfide bond.

Aspect 26. The multimeric polypeptide of aspect 25, wherein the firstMHC polypeptide or a linker between the epitope and the first MHCpolypeptide comprises an amino acid substitution to provide a first Cysresidue, and the second MHC polypeptide comprises an amino acidsubstitution to provide a second Cys residue, and wherein the disulfidelinkage is between the first and the second Cys residues.

Aspect 27. The multimeric polypeptide of any one of aspects 7-26,comprising a linker interposed between the epitope and the first MHCpolypeptide.

Aspect 28. The multimeric polypeptide of any one of aspects 7-26,comprising a linker interposed between the MHC polypeptide and theimmunomodulatory polypeptide.

Aspect 29. The multimeric polypeptide of any one of aspects 7-28,comprising 2 variant IL-2 polypeptides.

Aspect 30. The multimeric polypeptide of any one of aspects 8-28,comprising 3 variant IL-2 polypeptides.

Aspect 31. The multimeric polypeptide of aspect 29 or aspect 30, whereinthe 2 or 3 variant IL-2 polypeptides are in tandem, and wherein themultimeric polypeptide comprises a linker between the variant IL-2polypeptides.

Aspect 32. The multimeric polypeptide of any one of aspects 8-31,wherein the variant IL-2 comprises a substitution of one or more of E15,H16, D20, F42, Y45, and Q126/

Aspect 33 The multimeric polypeptide of any one of aspects 8-32, whereinthe variant IL-2 comprises a substitution of F42 with Ala, Gly, Val,Ile, or Leu.

Aspect 34. The multimeric polypeptide of aspect 33, wherein the variantIL-2 comprises substitutions of F42 and D20, or substitutions of F42 andH16.

Aspect 35. The multimeric polypeptide of aspect 33, wherein the variantIL-2 comprises substitutions of F42, D20, and Y45; or wherein thevariant IL-2 comprising substitutions of F42, H16, and Q126.

Aspect 36. A nucleic acid comprising a nucleotide sequence encoding arecombinant polypeptide,

i) wherein the recombinant polypeptide comprises, in order fromN-terminus to C-terminus;

a) an epitope;

b) a first major histocompatibility complex (MHC) polypeptide:

c) an immunomodulatory polypeptide;

d) a proteolytically cleavable linker or a ribosome skipping signal;

e) a second MHC polypeptide; and

f) an immunoglobulin (Ig) Fc polypeptide;

wherein the immunomodulatory polypeptide is a variant of a naturallyoccurring costimulatory protein, and wherein the variant exhibits areduced affinity for its counterpart costimulatory protein as comparedto the affinity of the naturally occurring costimulatory protein for thecounterpart costimulatory protein; or

ii) wherein the recombinant polypeptide comprises, in order fromN-terminus to C-terminus;

a) an epitope;

b) a first MHC polypeptide;

c) a proteolytically cleavable linker or a ribosome skipping signal;

d) an immunomodulatory polypeptide

e) a second MHC polypeptide; and

f) an Ig Fc polypeptide,

wherein the immunomodulatory polypeptide is a variant of a naturallyoccurring costimulatory protein, and wherein the variant exhibits areduced affinity for its counterpart costimulatory protein as comparedto the affinity of the naturally occurring costimulatory protein for thecounterpart costimulatory protein.

Aspect 37. A nucleic acid comprising a nucleotide sequence encoding arecombinant polypeptide,

i) wherein the recombinant polypeptide comprises, in order fromN-terminus to C-terminus;

a) an epitope;

b) a first major histocompatibility complex (MHC) polypeptide;

c) an immunomodulatory polypeptide:

d) a proteolytically cleavable linker or a ribosome skipping signal;

c) a second MHC polypeptide; and

f) an immunoglobulin (Ig) Fc polypeptide

wherein the immunomodulatory polypeptide is a variant immunomodulatorypolypeptide of any one of aspects 1-6; or

ii) wherein the recombinant polypeptide comprises, in order fromN-terminus to C-terminus;

a) an epitope;

b) a first MHC polypeptide;

c) a proteolytically cleavable linker or a ribosome skipping signal;

d) an immunomodulatory polypeptide

e) a second MHC polypeptide; and

f) an Ig Fc polypeptide,

wherein the immunomodulatory polypeptide is a variant immunomodulatorypolypeptide of any one of aspects 1-6.

Aspect 38. The nucleic acid of aspect 36 or 37, wherein the first MHCpolypeptide is a β2-microglobulin polypeptide; and wherein the secondMHC polypeptide is an MHC class I heavy chain polypeptide.

Aspect 39. The nucleic acid of aspect 38, wherein the β2-microglobulinpolypeptide comprises an amino acid sequence having at least 85% aminoacid sequence identity to one of the amino acid sequences set forth inFIG. 6.

Aspect 40. The nucleic acid of aspect 38, wherein the MHC class I heavychain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain.

Aspect 41. The nucleic acid of aspect 40, wherein the MHC class I heavychain polypeptide comprises an amino acid sequence having at least 85%amino acid sequence identity to the amino acid sequence set forth in anyone of FIG. 5A-5C.

Aspect 42. The nucleic acid of aspect 36 or 37, wherein the first MHCpolypeptide is an MHC Class II alpha chain polypeptide; and wherein thesecond MHC polypeptide is an MHC class 11 beta chain polypeptide.

Aspect 43. The nucleic acid of any one of aspects 36-42, wherein theepitope is a T-cell epitope.

Aspect 44. The nucleic acid of any one of aspects 36-43, wherein the IgFc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, anIgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, oran IμM Fc polypeptide.

Aspect 45. The nucleic acid of aspect 44, wherein the Ig Fc polypeptidecomprises an amino acid sequence having at least 85% amino acid sequenceidentity to an amino acid sequence depicted in FIGS. 4A-4C.

Aspect 46. The nucleic acid of any one of aspects 37-45, wherein thevariant IL-2 immunomodulatory polypeptide comprises a substitution ofone or more of E15, H16, D20, F42, Y45, and Q126.

Aspect 47. The nucleic acid of any one of aspects 36-46, wherein themultimeric polypeptide comprises a second immunomodulatory polypeptideselected from a CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, and HVEM.

Aspect 48. The nucleic acid of any one of aspects 36-47, wherein theproteolytically cleavable linker or ribosome skipping signal comprisesan amino acid sequence selected from:

a) LEVLFQGP (SEQ ID NO:29);

b) ENLYTQS (SEQ ID NO:30);

c) a furin cleavage site;

d) LVPR (SEQ ID NO:32);

e) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:33);

f) GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:34);

g) GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:35); and

h) GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:36).

Aspect 49. The nucleic acid of aspect 36-48, wherein the recombinantpolypeptide comprises, in order from N-terminus to C-terminus;

a) a first leader peptide:

b) the epitope;

c) the first MHC polypeptide;

d) the immunomodulatory polypeptide;

e) the proteolytically cleavable linker or ribosome skipping signal;

f) a second leader peptide;

g) the second MHC polypeptide; and

h) the immunoglobulin (Ig) Fc polypeptide.

Aspect 50. The nucleic acid of aspect 49, wherein the first leaderpeptide and the second leader peptide is a β2-M leader peptide.

Aspect 51. The nucleic acid of any one of aspects 36-50, wherein thenucleotide sequence is operably linked to a transcriptional controlelement.

Aspect 52. The nucleic acid of aspect 51, wherein the transcriptionalcontrol element is a promoter that is functional in a eukaryotic cell.

Aspect 53. The nucleic acid of any one of aspects 36-52, wherein thefirst MHC polypeptide or a linker between the epitope and the first MHCpolypeptide comprises an amino acid substitution to provide a first Cysresidue, and the second MHC polypeptide comprises an amino acidsubstitution to provide a second Cys residue, and wherein the first andthe second Cys residues provide for a disulfide linkage between thefirst MHC polypeptide and the second MHC polypeptide.

Aspect 54. A recombinant expression vector comprising the nucleic acidof any one of aspects 36-52, and wherein the vector is optionally aviral vector or a non-viral vector.

Aspect 55. A host cell genetically modified with the recombinantexpression vector of aspect 54.

Aspect 56. The host cell of aspect 55, wherein the host cell is invitro, and wherein the host cell is optionally genetically modified suchthat the cell does not produce an endogenous MHC β2-microglobulinpolypeptide.

Aspect 57. A composition comprising:

a) a first nucleic acid comprising a nucleotide sequence encoding afirst polypeptide comprising, in order from N-terminus to C-terminus;

-   -   i) an epitope;    -   ii) a first MHC polypeptide; and    -   iii) an immunomodulatory domain,

wherein the immunomodulatory polypeptide is a variant of a naturallyoccurring costimulatory protein, and wherein the variant exhibits areduced affinity for its counterpart costimulatory protein as comparedto the affinity of the naturally occurring costimulatory protein for thecounterpart costimulatory protein; and

b) a first nucleic acid comprising a nucleotide sequence encoding asecond polypeptide comprising, in order from N-terminus to C-terminus;

-   -   i) a second MHC polypeptide; and    -   ii) an Ig Fc polypeptide.

Aspect 58. A composition comprising:

a) a first nucleic acid comprising a nucleotide sequence encoding afirst polypeptide comprising, in order from N-terminus to C-terminus;

-   -   i) an epitope; and    -   ii) a first MHC polypeptide; and

b) a first nucleic acid comprising a nucleotide sequence encoding asecond polypeptide comprising, in order from N-terminus to C-terminus;

-   -   i) an immunomodulatory domain, wherein the immunomodulatory        domain is a variant of a naturally occurring costimulatory        protein, and wherein the variant exhibits a reduced affinity for        its counterpart costimulatory protein as compared to the        affinity of the naturally occurring costimulatory protein for        the counterpart costimulatory protein;    -   ii) a second MHC polypeptide; and    -   iii) an Ig Fc polypeptide.

Aspect 59. A composition comprising:

a) a first nucleic acid comprising a nucleotide sequence encoding afirst polypeptide comprising, in order from N-terminus to C-terminus;

-   -   i) an epitope;    -   ii) a first MHC polypeptide; and    -   iii) an immunomodulatory domain,

wherein the immunomodulatory domain is a variant IL-2 polypeptide of anyone of aspects 1-6; and

b) a first nucleic acid comprising a nucleotide sequence encoding asecond polypeptide comprising, in order from N-terminus to C-terminus;

-   -   i) a second MHC polypeptide; and    -   ii) an Ig Fc polypeptide.

Aspect 60. A composition comprising:

a) a first nucleic acid comprising a nucleotide sequence encoding afirst polypeptide comprising, in order from N-terminus to C-terminus;

-   -   i) an epitope; and    -   ii) a first MHC polypeptide; and

b) a first nucleic acid comprising a nucleotide sequence encoding asecond polypeptide comprising, in order from N-terminus to C-terminus;

-   -   i) an immunomodulatory domain, wherein the immunomodulatory        domain is a variant IL-2 polypeptide of any one of aspects 1-6;    -   ii) a second MHC polypeptide; and    -   iii) an Ig Fc polypeptide.

Aspect 61. The composition of any one of aspects 57-60, wherein thefirst and/or the second nucleic acid is present in a recombinantexpression vector.

Aspect 62. A host cell genetically modified with the composition of anyone of aspects 57-61.

Aspect 63. A method of producing the multimeric polypeptide of any oneof aspects 7-36, the method comprising:

a) culturing the host cell of any one of aspects 55, 56, and 62 in vitroin a culture medium under conditions such that the host cell synthesizesthe multimeric polypeptide; and

b) isolating the multimeric polypeptide from the host cell and/or fromthe culture medium.

Aspect 64. The method of aspect 63, wherein the second polypeptidecomprises an affinity tag, and wherein said isolating comprisescontacting the multimeric polypeptide produced by the cell with abinding partner for the affinity tag, wherein the binding partner isimmobilized, thereby immobilizing the multimeric polypeptide.

Aspect 65. The method of aspect 64, comprising eluting the immobilizedmultimeric polypeptide.

Aspect 66. A method of selectively activating an epitope-specific Tcell, the method comprising contacting the T cell with the multimericpolypeptide of any one of aspects 7-35, wherein said contactingselectively activates the epitope-specific T cell.

Aspect 67. The method of aspect 66, wherein said contacting is in vitro.

Aspect 68. The method of aspect 66, wherein said contacting is in vivo.

Aspect 69. The method of aspect 66, wherein the epitope is acancer-associated epitope, and wherein said administering selectivelyincreases the activity of a T cell specific for the cancer-associateepitope.

Aspect 70. A method of treating cancer in an individual, the methodcomprising administering to the individual an effective amount of:

a) the multimeric polypeptide of any one of aspects 7-35; or

b) one or more recombinant expression vectors comprising nucleotidesequences encoding the multimeric polypeptide of any one of aspects7-35; or

c) one or more mRNAs comprising nucleotide sequences encoding themultimeric polypeptide of any one of aspects 7-35,

wherein the epitope is a cancer-associated epitope, and wherein saidadministering effective to selectively activate a cancerepitope-specific T cell in an individual.

Aspect 71. The method of aspect 70, wherein said administering issubcutaneous.

Aspect 72. The method of aspect 70, wherein said administering isintravenous.

Aspect 73. The method of aspect 70, wherein said administering isperitumoral.

Aspect 74. The method of aspect 70, wherein said administering issystemic.

Aspect 75. The method of aspect 70, wherein said administering is distalto a treatment site.

Aspect 76. The method of aspect 70, wherein said administering is local.

Aspect 77. The method of aspect 70, wherein said administering is at ornear a treatment site.

Aspect 78. A composition comprising:

a) the multimeric polypeptide of any one of aspects 7-35; and

b) a pharmaceutically acceptable excipient.

Aspect 79. A composition comprising:

a) the nucleic acid of any one of aspects 36-53 or the recombinantexpression vector of aspect 54; and

b) a pharmaceutically acceptable excipient.

Aspect 80. A multimeric polypeptide comprising:

a) a first polypeptide comprising, in order from N-terminus toC-terminus;

i) an epitope;

ii) a β2-microglobulin (fβ2M) polypeptide comprising the amino acidsequence depicted in FIG. 34A; and

b) a second polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) a variant of a naturally occurring costimulatory protein, and        wherein the variant exhibits a reduced affinity for its        counterpart costimulatory protein as compared to the affinity of        the naturally occurring costimulatory protein for the        counterpart costimulatory protein, which variant optionally may        be a variant IL-2 polypeptide of any one of aspects 1-6;    -   ii) a major histocompatibility compels (MHC) heavy chain        polypeptide comprising the amino acid sequence depicted in FIG.        34C; and    -   iii) an IgG1 Fc polypeptide comprising one or more amino acid        substitutions selected from N297A, L234A, L235A, L234F, 1235E,        and P331S (N77A, L14A, L15A, L14F, L15E, and P111S,        respectively, based on the amino acid numbering depicted in FIG.        33A).

Aspect 81. The multimeric polypeptide of aspect 80, wherein the IgG1 Fcpolypeptide comprises an N297A substitution (N77A based on the aminoacid numbering depicted in FIG. 33A).

Aspect 82. The multimeric polypeptide of aspect 80, wherein the IgG1 Fepolypeptide comprises an L234A substitution and an L235A substitution(L14A and LISA based on the amino acid numbering depicted in FIG. 33A).

Aspect 83. The multimeric polypeptide of aspect 80, wherein the IgG1 Fcpolypeptide comprises an L234F substitution and an L235E substitution(L14F and L15E based on the amino acid numbering depicted in FIG. 33A).

Aspect 84. The multimeric polypeptide of aspect 80, wherein the IgG1 Fcpolypeptide comprises an L234F substitution, an L235E substitution, anda P331S (L14F, L15E, and P111S substitutions based on the amino acidnumbering depicted in FIG. 33A).

Aspect 85. The multimeric polypeptide of any one of aspects 80-84,wherein the second polypeptide comprises two copies of the variant IL-2polypeptide.

Aspect 86. The multimeric polypeptide of any one of aspects 80-85,wherein the first polypeptide comprises a peptide linker between theepitope and the β2M polypeptide.

Aspect 87. The multimeric polypeptide of any one of aspects 80-86,wherein the second polypeptide comprises a peptide linker between one ormore of:

a) a first copy of the variant IL-2 polypeptide and a second copy of thevariant IL-2 polypeptide;

b) the variant IL-2 polypeptide and the MHC heavy chain polypeptide; and

c) between the MHC heavy chain polypeptide and the IgG Fc polypeptide.

Aspect 88. The multimeric polypeptide of aspect 86 or aspect 87, whereinthe peptide linker is selected from (GGGGS)₃, (GGGGS)₄, and AAAGG.

Aspect 89. A multimeric polypeptide comprising:

a) a first polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) an epitope;    -   ii) a β2-microglobulin polypeptide comprising the amino acid        sequence depicted in FIG. 34A; and

b) a second polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) a variant IL-2 polypeptide comprising the amino acid sequence        depicted in FIG. 34B;    -   ii) a major histocompatibility comples (MHC) heavy chain        polypeptide comprising the amino acid sequence depicted in FIG.        34C; and    -   iii) an IgG1 Fc polypeptide comprising one or more amino acid        substitutions selected from N297A, L234A, L235A, L234F, L235E,        and P331S (N77A, L14A, L15A, L14F, L15E, and P111 S,        respectively, based on the amino acid numbering depicted in FIG.        33A).

Aspect 90. The multimeric polypeptide of aspect 89, wherein the IgG1 Fcpolypeptide comprises an N297A substitution (N77A based on the aminoacid numbering depicted in FIG. 33A).

Aspect 91. The multimeric polypeptide of aspect 89, wherein the IgG1 Fcpolypeptide comprises an L234A substitution and an L235A substitution(L14A and LISA based on the amino acid numbering depicted in FIG. 33A).

Aspect 92. The multimeric polypeptide of aspect 89, wherein the IgG1 Fepolypeptide comprises an L234F substitution and an L235E substitution(L14F and L15E based on the amino acid numbering depicted in FIG. 33A).

Aspect 93. The multimeric polypeptide of aspect 89, wherein the IgG1 Fcpolypeptide comprises an L234F substitution, an L235E substitution, anda P331S substitution (L14F, L15E, and P111S based on the amino acidnumbering depicted in FIG. 33A).

Aspect 94. The multimeric polypeptide of any one of aspects 89-93,wherein the second polypeptide comprises two copies of the variant IL-2polypeptide.

Aspect 95. The multimeric polypeptide of any one of aspects 89-94,wherein the first polypeptide comprises a peptide linker between theepitope and the β2M polypeptide.

Aspect 96. The multimeric polypeptide of any one of aspects 89-95,wherein the second polypeptide comprises a peptide linker between one ormore of:

a) a first copy of the variant IL-2 polypeptide and a second copy of thevariant IL-2 polypeptide;

b) the variant IL-2 polypeptide and the MHC heavy chain polypeptide; and

c) the MHC heavy chain polypeptide and the IgG1 Fc polypeptide.

Aspect 97. The multimeric polypeptide of aspect 95 or aspect 96, whereinthe peptide linker is selected from (GGGGS)₃, (GGGGS)₄, and AAAGG.

Aspect 98. A multimeric polypeptide comprising:

a) a first polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) an epitope comprising the amino acid sequence YMLDLQPETT (SEQ        ID NO:13);    -   ii) a β2-microglobulin polypeptide comprising the amino acid        sequence depicted in FIG. 34A; and

b) a second polypeptide comprising, in order from N-terminus toC-terminus;

-   -   i) a variant IL-2 polypeptide comprising the amino acid sequence        depicted in FIG. 34B;    -   ii) a major histocompatibility comples (MHC) heavy chain        polypeptide comprising the amino acid sequence depicted in FIG.        34C; and    -   iii) an IgG1 Fc polypeptide comprising the amino acid sequence        depicted in FIG. 33A, 33B, 33C, or 33D.

Aspect 99. The multimeric polypeptide of aspect 98, wherein the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33B.

Aspect 100. The multimeric polypeptide of aspect 98, wherein the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33C.

Aspect 101. The multimeric polypeptide of aspect 98, wherein the IgG1 Fcpolypeptide comprises the amino acid sequence depicted in FIG. 33D.

Aspect 102. The multimeric polypeptide of any one of aspects 98-101,wherein the second polypeptide comprises two copies of the variant IL-2polypeptide.

Aspect 103. The multimeric polypeptide of any one of aspects 98-102,wherein the first polypeptide comprises a peptide linker between theepitope and the fβ2M polypeptide.

Aspect 104. The multimeric polypeptide of any one of aspects 98-103,wherein the second polypeptide comprises a peptide linker between one ormore of:

a) a first copy of the variant IL-2 polypeptide and a second copy of thevariant IL-2 polypeptide;

b) the variant IL-2 polypeptide and the MHC heavy chain polypeptide; and

c) the MHC heavy chain polypeptide and the IgG1 Fc polypeptide.

Aspect 105. The multimeric polypeptide of aspect 103 or aspect 104,wherein the peptide linker is selected from (GGGGS), (GGGGS)₄, andAAAGG.

Aspect 106. A multimeric polypeptide comprising:

a) a first polypeptide comprising the amino acid sequence depicted inFIG. 31;

b) a second polypeptide comprising the amino acid sequence depicted inFIG. 22.

Aspect 107. A multimeric polypeptide comprising:

a) a first polypeptide comprising the amino acid sequence depicted inFIG. 31;

h) a second polypeptide comprising the amino acid sequence depicted inFIG. 25.

Aspect 108. A multimeric polypeptide comprising:

a) a first polypeptide comprising the amino acid sequence depicted inFIG. 31;

b) a second polypeptide comprising the amino acid sequence depicted inFIG. 28.

Aspect 109. A pharmaceutical composition comprising:

a) a multimeric polypeptide according to any one of aspects 80-108; and

b) a pharmaceutically acceptable excipient.

Aspect 110. One or more nucleic acids comprising nucleotide sequencesencoding the first and/or the second polypeptide of the multimericpolypeptide according to any one of aspects 80-108.

Aspect 111. The one or more nucleic acids of aspect 110, wherein thenucleic acid(s) is/are present in recombinant expression vectors.

Aspect 112. A method of selectively activating an epitope-specific Tcell, the method comprising contacting the T cell with the multimericpolypeptide of any one of aspects 80-108, wherein said contactingselectively activates the epitope-specific T cell.

Aspect 113. The method of aspect 112, wherein said contacting is invitro.

Aspect 114. The method of aspect 112, wherein said contacting is invivo.

Aspect 115. A method comprising administering to an individual aneffective amount of:

a) the multimeric polypeptide of any one of aspects 80-108; or

b) one or more recombinant expression vectors comprising nucleotidesequences encoding the multimeric polypeptide of any one of aspects80-108; or

c) one or more mRNAs comprising nucleotide sequences encoding themultimeric polypeptide of any one of aspects 80-108, wherein saidadministering induces a T cell response to epitope in the individual.

Aspect 116. The method of aspect 115, wherein said administering issubcutaneous.

Aspect 117. The method of aspect 115, wherein said administering isintravenous.

Aspect 118. The method of aspect 115, wherein said administering issystemic.

Aspect 119. The method of aspect 115, wherein said administering isintramuscular.

Aspect 120. The method of aspect 115, wherein said administering isdistal to a treatment site.

Aspect 121. The method of aspect 115, wherein said administering islocal.

Aspect 122. The method of aspect 115, wherein said administering is ator near a treatment site.

Aspect 123. A method of delivering a costimulatory polypeptideselectively to target T cell, the method comprising contacting a mixedpopulation of T cells with a multimeric polypeptide of any one ofaspects 7-35 and 80-108, wherein the mixed population of T cellscomprises the target T cell and non-target T cells, wherein the target Tcell is specific for the epitope present within the multimericpolypeptide, and wherein said contacting delivers the costimulatorypolypeptide present within the multimeric polypeptide to the target Tcell.

Aspect 124. A method of delivering IL-2 or an IL-2 variant selectivelyto a target T cell, the method comprising contacting a mixed populationof T cells with the multimeric polypeptide of any one of aspects 8-35and 80-108, wherein the mixed population of T cells comprises the targetT cell and non-target T cells, wherein the target T cell is specific forthe epitope present within the multimeric polypeptide, and wherein saidcontacting delivers the IL-2 or IL-2 variant present within themultimeric polypeptide to the target T cell.

Aspect 125. The method of aspect 123 or 124, wherein the population of Tcells is in vitro.

Aspect 126. The method of aspect 123 or 124, wherein the population of Tcells is in vivo in an individual.

Aspect 127. The method of aspect 126, comprising administering themultimeric polypeptide to the individual.

Aspect 128. The method of any one of aspects 123-127, wherein the targetT cell is a regulatory T cell.

Aspect 129. The method of any one of aspects 123-127, wherein the targetT cell is a cytotoxic T cell.

Aspect 130. The method of aspect 123 or 124, wherein the mixedpopulation of T cells is an in vitro population of mixed T cellsobtained from an individual, and wherein said contacting results inactivation and/or proliferation of the target T cell, generating apopulation of activated and/or proliferated target T cells.

Aspect 131. The method of claim 130, further comprising administeringthe population of activated and/or proliferated target T cells to theindividual.

Aspect 132. A method of detecting, in a mixed population of T cellsobtained from an individual, the presence of a target T cell that bindsan epitope of interest, the method comprising: a) contacting in vitrothe mixed population of T cells with the multimeric polypeptide of anyone of claims 7-35 and 80-108, wherein the multimeric polypeptidecomprises the epitope of interest; and b) detecting activation and/orproliferation of T cells in response to said contacting, whereinactivated and/or proliferated T cells indicates the presence of thetarget T cell.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min. minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt. nucleotide(s); kiloDalton(s), kDa;i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

Example 1: Production of IL-2/synTac

Production of IL-2/synTac by transiently transfected mammalian cells wasanalyzed. As shown in FIG. 7A, production levels (in mg/L culturemedium) of two different IL-2/synTacs, 6-7 days following transienttransfection of the cells, was greater than 90 mg/L.

The IL-2/synTacs produced by the mammalian cells was purified, andsubjected to reducing and non-reducing polyacrylamide gelelectrophoresis. The results are depicted in FIG. 7B. Sizes are given inkDa.

IL-2/synTacs were generated, in which the IL-2 polypeptide was in the“light chain” (i.e., the polypeptide comprising MHC Class I light chain;e.g., fβ2M) or in the “heavy chain” (i.e., the polypeptide comprisingMHC Class I heavy chain). Expression levels and stability of theIL-2/synTacs were analyzed.

The synTacs were produced in mammalian cells. As shown in FIG. 8A, theIL-2/synTac comprising IL-2 on the heavy chain was produced at levelsabout 25-fold higher than the level of the IL-2/synTac comprising IL-2on the light chain.

The IL-2/synTacs produced by mammalian cells were subjected to reducingand non-reducing polyacrylamide gel electrophoresis; and the gels werestained with Coomassie blue. As shown in FIG. 8B, the IL-2/synTaccomprising IL-2 on the heavy chain was more stable than the IL-2/synTaccomprising IL-2 on the light chain. Sizes are given in kDa.

Expression levels of IL-2/synTacs comprising variant IL-2 were assessed.FIG. 9 depicts the expression level of IL-2/syn-Tacs, in which the IL-2is wild-type (wt), or comprises various combinations of F42A, D20K,Q126A, E15A, Y45A, and H16A. The expression levels are expressed aspercent change relative to expression levels of a synTac with wild-typeIL-2.

The effect of the copy number of IL-2 in an IL-2/synTac on expressionlevels was evaluated, IL-2/synTacs comprising one copy (1×), two copies(2×) or three copies (3×) in the synTac. The various TL-2/synTacs wereproduced in mammalian cells, and expression levels were assayed. Thedata are depicted in FIG. 10. IL-2/synTacs with one or two copies ofIL-2 exhibit similar expression levels, while an IL-2/synTac with threecopies of IL-2 exhibited lower expression levels. Expression levels areexpressed as fold change relative to the expression level of theIL-2/synTac with a single copy of IL-2.

Example 2: In Vitro Activity of IL-2/synTac

To achieve maximal specificity of targeting through a T-cell receptor,the affinity of the co-stimulatory polypeptide for its ligand should belower than the affinity of MHC for the TCR. The peptide/MHC affinity forTCR can be about 10 μM.

An IL-2/synTac was generated, comprising two copies of a variant IL-2comprising F42A and H16A substitutions. Costimulatory signaling inducedby the IL-2/synTac was tested on antigen-specific CD8⁺ T cells andnon-specific CD8⁺ T cells. Antigen-specific CD8⁺ T cells andnon-specific CD8⁺ T cells were contacted with various concentrations ofthe IL-2/synTac.

As shown in FIG. 11, the IL-2/synTac induced costimulatory signaling inantigen-specific CD8⁺ T cells at a much lower concentration than innon-specific CD8⁺ T cells.

Selectivity of IL-2/synTac binding was tested, CD8⁺ T cells wereisolated from spleens of LCMV or OT1 mice. The CD8⁺ T cells wereincubated with IL-2/synTacs at various concentrations, and allowed tobind for 20 minutes. The IL-2/synTacs comprise IgG2a Fc. Binding ofIL-2/synTacs to the CD8⁺ T cells was detected using phycoerythrin(PE)-labeled anti-IgG2a antibody. PE fluorescence was detected usingflow cytometry to determine the percent of cells bound to IL-2/synTac.

As shown in FIG. 12, IL-2/synTac binds in an antigen-specific manner toLCMV CD8⁺ T cells, but does not exhibit significant binding to OT1 CD8⁺T cells. Thus, IL-2/synTac selectively binds to CD8⁺ T cells specificfor the epitope present in the IL-2/synTac.

It was determined whether an IL-2/synTac selectively activates target Tcells, CD8⁺ T cells were isolated from spleens of LCMV or OT1 mice. TheIL-2/synTacs used included either the F42A single amino acidsubstitution, or the F42A and H16A substitutions. The CD8⁺ T cells werestimulated with IL-2/synTacs at various concentrations for 20 minutes.The cells were then stained with PE-labelled anti-phospho-STAT5antibody. PE fluorescence was detected using flow cytometry to determinethe percent of cells that are phospho-STAT5 positive, wherephospho-STAT5 is a marker of activation.

As shown in FIG. 13, IL-2/synTac induced CD8⁺ stimulation (as indicatedby the % phospho-STAT5-positive cells) in antigen-specific (LCMV) CD8⁺ Tcells at much lower concentrations than in non-specific (BL6) CD8⁺ Tcells.

The specific activity of various IL-2/synTacs was analyzed. IL-2/synTacscomprising a single copy of IL-2, two copies of IL-2, or three copies ofIL-2, where the IL-2 comprised various combinations of F42A, D20K,Q126A, E15A, H16A, and Y45A substitutions, were tested at variousconcentrations for stimulation of CD8⁺ antigen-specific (LCMV) ornon-specific (BL6) cells. The percent phospho-signal transducer andactivator of transcription 5 (pSTATS)-positive was determined. The dataare depicted in FIG. 14A-14F.

Example 3: In Vivo Activity of IL-2/synTac

The in vivo activity of IL-2/synTac was tested. The in vivo fold changein antigen-specific CD8⁺ T cells was tested, following administration ofphosphate buffered saline (PBS), recombinant IL-2 (rIL-2), or anIL-2/synTac of the present disclosure. The data are shown in FIG. 15,left panel. The data indicate that IL-2/synTac is 10 times more potentthan rIL-2.

The in vivo specificity of IL-2/synTac was tested. Antigen-specific andnon-antigen-specific responses following administration of PBS, rIL-2,or IL-2/synTac was assessed. The data are expressed as percent of lymphnode cells that were antigen-specific or antigen non-specific followingadministration of PBS, rIL-2, or IL-2/synTac. As depicted in FIG. 15,right panel, IL-2/synTac induced an antigen-specific response (expressedas % maximum dilution of carboxyfluorescein succinimidyl ester (CFSE),an index of T cell proliferation). In contrast, the response induced byrIL-2 was not antigen-specific.

A dose response assay was conducted, IL-2/synTac (F42A, H16A) wasadministered intraperitoneally at concentrations of 4 mg/kg, 8 mg/kg,and 16 mg/kg. The results are shown in FIG. 16A. As shown in FIG. 16A,IL-2/synTac administered at 4 mg/kg or 8 mg/kg gave similar results;IL-2/synTac administered at 16 mg/kg induced the most potentimmunostimulatory activity.

The effect of route of administration of IL-2/synTac was tested,IL-2/synTac (F42A, H16A) was administered at 4 mg/kg, eithersubcutaneously (SubQ) or intraperitoneally (IP). As shown in FIG. 16B,subcutaneous administration resulted in a more potent immunostimulatoryactivity than IP administration.

The effect of IL-2 copy number on efficacy was determined, IL-2/synTacscomprising a single copy of IL-2 (F42A, H16A) or two copies of IL-2(F42A, H16A) were injected into mice with tumors bearing an HPV E7epitope. The epitope included in the IL-2/synTacs was the HPV E7epitope. As shown in FIGS. 17A and 17B, an IL-2/synTac comprising twocopies of IL-2(F42A, H16A) were more effective at reducing tumor sizethan an IL-2/synTac comprising only a single copy of IL-2(F42A, H16A).

Example 4: PK/PD and Stability Studies of IL-2/synTac

Pharmacokinetic (PK) analysis of IL-2/synTac was carried out,IL-2/synTac (F42A, D20K, H16A) was administered IP at 10 mg/kg. Atvarious time points post-administration, serum samples were obtained andthe level of IL-2/synTac was measured in the serum samples. As shown inFIG. 18, the serum half-life of the IL-2/synTac was about 4 hours.

IL-2/synTac was injected IP into a C57BL/6 mouse at 10 mg/kg, and serumwas collected two hours after injections. The IL-2/synTac included aHis₆ tag. 100 ng of the input protein, or the equivalent of 40 μl ofserum, was subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), and probed with an anti-(His)₆ antibody oran anti-β-2M antibody. The results, depicted in FIG. 19, show thatIL-2/synTac remains stable and intact for at least 2 hours in vivo.

IL-2/synTac was kept at 4° C., or 37° C. for 5 days, 0.5 mg of eachsample (at 10 mg/ml) was analyzed by size exclusion chromatography. Asshown in FIG. 20, IL-2/synTac is stable and intact for at least 5 daysat 4° C., or 37° C.

Example 5: IL-2/synTac-Mediated Expansion of Human CMV-Specific CD8⁺ TCells

Peripheral blood mononuclear cells (PBMCs) from human donors werescreened for reactivity towards a cytomegalovirus (CMV)-peptide poolusing an IFN-gamma enzyme-linked immunospot (ELISPOT) assay. The PBMCswere categorized by spot forming count (SFC) as high, medium, low, or noCMV-precursor groups. PBMCs from each group were stimulated with dosesof IL-2/synTac (“CUE:IL-2”; a synTac comprising 2 copies of a variantIL-2 MOD comprising H16A and F42A substitutions) ranging from 30 nM to 2nM. Fifty percent of the conditioned media was replaced with fresh mediaon day 5. On day 7, the samples were stained with a panel of antibodiesand analyzed by flow cytometry. Pentamer staining targeting the CMVpeptide NLVPMVATV (SEQ ID NO:37) was used to determine the frequency ofantigen-specific CD8⁺ cells. The data are presented in FIG. 35. The EC₅₀of IL-2/synTac was determined to be in the range of from about 1 nM toabout 5 nM. FIG. 35 shows the fold expansion of antigen-specific CD8⁺cells compared to untreated controls. Numerical values on the X-axisrepresent the SFC count of each donor PBMC. Error bars represent themean+/−SD values from the technical replicates of each data points.

The data shown in FIG. 35 indicate that an IL-2/synTac is effective toexpand the number of epitope-specific CD8⁺ T cells, where there is ameasurable (e.g., by pentamer staining or by SFC) precursor populationof such epitope-specific CD8⁺ T cells.

Example 6: IL-2/synTac with Amino Acid Substitutions at H16

IL-2/synTac variants were generated with substitutions at H16.Expression levels and affinity for IL-2R were determined. Affinity forIL-2 R was determined using BLI. The data are presented in FIG. 36.

Example 7: IL-2/synTac Effects on Primary Human Antigen-Specific CD8 TICells

A variant IL-2/synTac was contacted with primary CD8⁺ T cells from ahuman subject. The variant IL-2/synTac includes: i) HPV16 E7 (11-20)(YMLDLQPET; SEQ ID NO:13) as the epitope-presenting peptide; and ii) 2copies of a variant IL-2 MOD comprising H16A and F42A substitutions).Binding of the variant IL-2/synTac to CD8⁺ T cells specific for HPV16 E7(11-20), or to bulk CD8⁺ T cells was assessed. The data are shown inFIG. 37.

FIG. 37 depicts binding of a variant IL-2/synTac of the presentdisclosure to primary human HPV16 E7 (11-20)-specific CD8⁺ T cells, asdetected by flow cytometry. The EC, for binding to CD8⁺ T cells specificfor HPV16 E7 (11-20) was 2.6 nM. Thus, the variant IL-2/synTac exhibitedhigh-affinity interaction with tumor antigen-specific primary human Tcells. Binding was highly selective for antigen-specific T cells,compared to the binding to non-target (bulk) CD8⁺ T cells.

The effect of binding of the variant IL-2/synTac to primary human HPV16E7 (11-20)-specific CD8⁺ T cells on phosphorylation of the T-cellreceptor (TCR)-proximal marker SLP76 was assessed. The data are shown inFIG. 38.

FIG. 38 depicts the effect of binding of the variant IL-2/synTac toprimary human HPV16 E7 (11-20)-specific CD8⁺ T cells on phosphorylationof SLP76. Binding of the variant IL-2/synTac to primary human HPV16 E7(11-20)-specific CD8⁺ T cells resulted in a rapid increase inphosphorylation of SLP76. The effect was potent (EC₅₀=65 nM). The effectwas also selective, as a control IL-2/synTac that comprises a CMVpeptide instead of HPV16 E7 (11-20) resulted in only low levels of SLP76phosphorylation.

Key markers of T-cell activation and cytolytic activity were assessed.Primary human HPV16 E7 (11-20)-specific T cells were incubated for 2days with 0 nM or 100 nM variant IL-2/synTac. The variant IL-2/synTacincludes: i) HPV16 E7 (11-20) as the epitope-presenting peptide; and ii)2 copies of a variant IL-2 MOD comprising H16A and F42A substitutions).Production of: i) CD25, a marker of CD8⁺ T cell activation; ii) granzymeB, a key mediator of target cell death via the granule-mediated pathway;and iii) CD107α, a marker of degranulation on CD8⁺ T cells, wasassessed. The data are shown in FIG. 39.

FIG. 39 depicts the effect of binding of the variant IL-2synTac toprimary human HPV16 E7 (11-20)-specific T cells on production of CD25,granzyme B, and CD107α. The data show that binding of the variantIL-2/synTac to primary human HPV16 E7 (11-20)-specific T cells inducesdifferentiation of the T cells into cytolytic effector cells, asevidenced by the increased expression of CD25, granzyme B, and CD107α.

The effect of binding of the variant IL-2/synTac to primary human HPV16E7 (11-20)-specific CD8⁺ T cells on production of IFN-γ was assessed. AnELISpot assay was used to detect IFN-γ production. The data are shown inFIG. 40.

FIG. 40 depicts the effect of binding of the variant IL-2/synTac toprimary human HPV16 E7 (11-20)-specific CD8⁺ T cells on production ofIFN-γ. The data show that binding of the variant IL-2/synTac to primaryhuman HPV16 E7 (11-20)-specific CD8⁺ T cells resulted in adose-dependent secretion of IFN-γ. No IFN-γ production was observed witha control IL-2/synTac that comprises a CMV peptide instead of HPV16 E7(1-20).

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-132. (canceled)
 133. A molecule comprising: a) a first polypeptidecomprising: i) an HPV16 cancer-associated peptide epitope; and ii) firstclass I major histocompatibility complex (MHC) polypeptide; b) a secondpolypeptide comprising a second class I MHC polypeptide; and c) one ormore immunomodulatory polypeptides, wherein the first and secondpolypeptide form a heterodimer, wherein the first and/or the secondpolypeptide comprises the one or more immunomodulatory polypeptides,wherein the one or more immunomodulatory polypeptides comprises one ormore variant interleukin-2 (IL-2) polypeptides comprising an amino acidsequence having at least 85% amino acid sequence identity to set forthin SEQ ID NO:1, and having one or more amino acid substitutions relativeto set forth in SEQ ID NO:1, and wherein at least one of the one or moreimmunomodulatory polypeptides that is a variant IL-2 polypeptideexhibits reduced binding affinity to an IL-2 receptor (IL2R) comprisingalpha, beta, and gamma polypeptides having amino acid sequences setforth in SEQ ID NOs:54-56, compared to the binding affinity of a controlIL-2 polypeptide comprising the IL-2 amino acid sequence set forth inSEQ ID NO:1 for the IL2R polypeptide.
 134. A composition comprising oneor more nucleic acids encoding the first and second polypeptides ofclaim
 133. 135. A method of producing the molecule of claim 133, themethod comprising culturing a host cell comprising a compositionaccording to claim 134 in vitro in a culture medium under conditionssuch that the host cell produces the molecule.
 136. A method ofactivating an epitope-specific T cell, the method comprising contactingin vivo the T cell with the molecule of claim 133, wherein saidcontacting activates the epitope-specific T cell.
 137. A method ofactivating an epitope-specific T cell, the method comprising contactingex vivo the T cell with the molecule of claim 133, wherein saidcontacting activates the epitope-specific T cell.
 138. A method oftreating cancer in an individual, the method comprising administering tothe individual an effective amount of the molecule of claim
 133. 139. Amethod of detecting, in a mixed population of T cells obtained from anindividual, the presence of a target T cell that binds an epitope ofinterest, the method comprising: a) contacting in vitro the mixedpopulation of T cells with the molecule of claim 133, wherein themolecule comprises the epitope of interest; and b) detecting activationand/or proliferation of target T cells in response to said contacting.140. A molecule comprising: a) a first polypeptide comprising: i) acancer-associated peptide epitope other than an HPV16 cancer-associatedepitope; and ii) first class I major histocompatibility complex (MHC)polypeptide; b) a second polypeptide comprising a second class I MHCpolypeptide; and c) one or more immunomodulatory polypeptides, whereinthe first and second polypeptide form a heterodimer, wherein the firstand/or the second polypeptide comprises the one or more immunomodulatorypolypeptides, wherein the one or more immunomodulatory polypeptidescomprises one or more variant interleukin-2 (IL-2) polypeptidescomprising an amino acid sequence having at least 85% amino acidsequence identity to set forth in SEQ ID NO:1, and having one or moreamino acid substitutions relative to set forth in SEQ ID NO:1, andwherein at least one of the one or more immunomodulatory polypeptidesthat is a variant IL-2 polypeptide exhibits reduced binding affinity toan IL-2 receptor (IL2R) comprising alpha, beta, and gamma polypeptideshaving amino acid sequences set forth in SEQ ID NOs:54-56, compared tothe binding affinity of a control IL2 polypeptide comprising the IL2amino acid sequence set forth in SEQ ID NO:1 for the IL2R polypeptide.141. A composition comprising one or more nucleic acids encoding thefirst and second polypeptides of claim
 140. 142. A method of producingthe molecule of claim 140, the method comprising culturing a host cellcomprising a composition according to claim 141 in vitro in a culturemedium under conditions such that the host cell produces the molecule.143. A method of activating an epitope-specific T cell, the methodcomprising contacting in vivo the T cell with the molecule of claim 140,wherein said contacting activates the epitope-specific T cell.
 144. Amethod of activating an epitope-specific T cell, the method comprisingcontacting ex vivo the T cell with the molecule of claim 140, whereinsaid contacting activates the epitope-specific T cell.
 145. A method oftreating cancer in an individual, the method comprising administering tothe individual an effective amount of the molecule of claim
 140. 146. Amethod of detecting, in a mixed population of T cells obtained from anindividual, the presence of a target T cell that binds an epitope ofinterest, the method comprising: a) contacting in vitro the mixedpopulation of T cells with the molecule of claim 140, wherein themolecule comprises the epitope of interest; and b) detecting activationand/or proliferation of target T cells in response to said contacting.147. A molecule comprising: a) a first polypeptide comprising: i) apeptide epitope present in a self antigen; and ii) first class I majorhistocompatibility complex (MHC) polypeptide; b) a second polypeptidecomprising a second class I MHC polypeptide; and c) one or moreimmunomodulatory polypeptides, wherein the first and second polypeptideform a heterodimer, wherein the first and/or the second polypeptidecomprises the one or more immunomodulatory polypeptides, wherein the oneor more immunomodulatory polypeptides comprises one or more variantinterleukin-2 (IL-2) polypeptides comprising an amino acid sequencehaving at least 85% amino acid sequence identity to set forth in SEQ IDNO:1, and having one or more amino acid substitutions relative to setforth in SEQ ID NO:1, and wherein at least one of the one or moreimmunomodulatory polypeptides that is a variant IL-2 polypeptideexhibits reduced binding affinity to an IL-2 receptor (IL2R) comprisingalpha, beta, and gamma polypeptides having amino acid sequences setforth in SEQ ID NOs:54-56, compared to the binding affinity of a controlIL-2 polypeptide comprising the IL-2 amino acid sequence set forth inSEQ ID NO:1 for the IL2R polypeptide.
 148. A molecule comprising: a) afirst polypeptide comprising: i) a pathogen-associated peptide epitope;and ii) first class I major histocompatibility complex (MHC)polypeptide; b) a second polypeptide comprising a second class I MHCpolypeptide; and c) one or more immunomodulatory polypeptides, whereinthe first and second polypeptide form a heterodimer, wherein the firstand/or the second polypeptide comprises the one or more immunomodulatorypolypeptides, wherein the one or more immunomodulatory polypeptidescomprises one or more variant interleukin-2 (IL-2) polypeptidescomprising an amino acid sequence having at least 85% amino acidsequence identity to set forth in SEQ ID NO:1, and having one or moreamino acid substitutions relative to set forth in SEQ ID NO:1, andwherein at least one of the one or more immunomodulatory polypeptidesthat is a variant IL-2 polypeptide exhibits reduced binding affinity toan IL-2 receptor (IL2R) comprising alpha, beta, and gamma polypeptideshaving amino acid sequences set forth in SEQ ID NOs:54-56, compared tothe binding affinity of a control IL-2 polypeptide comprising the IL-2amino acid sequence set forth in SEQ ID NO:1 for the IL2R polypeptide.149. A molecule comprising: a) a first polypeptide comprising: i) apeptide epitope other than an epitope of a cancer-associated antigen, aself-antigen, or a pathogen-associated epitope; and ii) first class Imajor histocompatibility complex (MHC) polypeptide; b) a secondpolypeptide comprising a second class I MHC polypeptide; and c) one ormore immunomodulatory polypeptides, wherein the first and secondpolypeptide form a heterodimer, wherein the first and/or the secondpolypeptide comprises the one or more immunomodulatory polypeptides,wherein the one or more immunomodulatory polypeptides comprises one ormore interleukin-2 (IL-2) polypeptides comprising an amino acid sequencehaving at least 85% amino acid sequence identity to set forth in SEQ IDNO:1, and having one or more amino acid substitutions relative to setforth in SEQ ID NO:1, and wherein at least one of the one or moreimmunomodulatory polypeptides that is a variant IL-2 polypeptideexhibits reduced binding affinity to an IL-2 receptor (IL2R) comprisingalpha, beta, and gamma polypeptides having amino acid sequences setforth in SEQ ID NOs:54-56, compared to the binding affinity of a controlIL-2 polypeptide comprising the IL-2 amino acid sequence set forth inSEQ ID NO:1 for the IL2R polypeptide.
 150. A molecule comprising: a) afirst polypeptide comprising: i) a cancer-associated peptide epitope;and ii) first class II major histocompatibility complex (MHC)polypeptide; b) a second polypeptide comprising a second class II MHCpolypeptide; and c) one or more immunomodulatory polypeptides, whereinthe first and second polypeptide form a heterodimer, wherein the firstand/or the second polypeptide comprises the one or more immunomodulatorypolypeptides, wherein the one or more immunomodulatory polypeptidescomprises one or more variant interleukin-2 (IL-2) polypeptidescomprising an amino acid sequence having at least 85% amino acidsequence identity to set forth in SEQ ID NO:1, and having one or moreamino acid substitutions relative to set forth in SEQ ID NO:1, andwherein at least one of the one or more immunomodulatory polypeptidesthat is a variant IL-2 polypeptide exhibits reduced binding affinity toan IL-2 receptor (IL2R) comprising alpha, beta, and gamma polypeptideshaving amino acid sequences set forth in SEQ ID NOs:54-56, compared tothe binding affinity of a control IL-2 polypeptide comprising the IL-2amino acid sequence set forth in SEQ ID NO:1 for the IL2R polypeptide.151. A molecule comprising: a) a first polypeptide comprising: i) apeptide epitope present in a self antigen; and ii) first class II majorhistocompatibility complex (MHC) polypeptide; b) a second polypeptidecomprising a second class II MHC polypeptide; and c) one or moreimmunomodulatory polypeptides, wherein the first and second polypeptideform a heterodimer, wherein the first and/or the second polypeptidecomprises the one or more immunomodulatory polypeptides, wherein the oneor more immunomodulatory polypeptides comprises one or more variantinterleukin-2 (IL-2) polypeptides comprising an amino acid sequencehaving at least 85% amino acid sequence identity to set forth in SEQ IDNO:1, and having one or more amino acid substitutions relative to setforth in SEQ ID NO:1, and wherein at least one of the one or moreimmunomodulatory polypeptides that is a variant IL-2 polypeptideexhibits reduced binding affinity to an IL-2 receptor (IL2R) comprisingalpha, beta, and gamma polypeptides having amino acid sequences setforth in SEQ ID NOs:54-56, compared to the binding affinity of a controlIL-2 polypeptide comprising the IL-2 amino acid sequence set forth inSEQ ID NO:1 for the IL2R polypeptide.
 152. A molecule comprising: a) afirst polypeptide comprising: i) a pathogen-associated peptide epitope;and ii) first class II major histocompatibility complex (MHC)polypeptide; b) a second polypeptide comprising a second class II MHCpolypeptide; and c) one or more immunomodulatory polypeptides, whereinthe first and second polypeptide form a heterodimer, wherein the firstand/or the second polypeptide comprises the one or more immunomodulatorypolypeptides, wherein the one or more immunomodulatory polypeptidescomprises one or more variant interleukin-2 (IL-2) polypeptidescomprising an amino acid sequence having at least 85% amino acidsequence identity to set forth in SEQ ID NO:1, and having one or moreamino acid substitutions relative to set forth in SEQ ID NO:1, andwherein at least one of the one or more immunomodulatory polypeptidesthat is a variant IL-2 polypeptide exhibits reduced binding affinity toan IL-2 receptor (IL2R) comprising alpha, beta, and gamma polypeptideshaving amino acid sequences set forth in SEQ ID NOs:54-56, compared tothe binding affinity of a control IL-2 polypeptide comprising the IL-2amino acid sequence set forth in SEQ ID NO:1 for the IL2R polypeptide.153. A molecule comprising: a) a first polypeptide comprising: i) apeptide epitope other than an epitope of a cancer-associated antigen, aself-antigen, or a pathogen-associated epitope; and ii) first class IImajor histocompatibility complex (MHC) polypeptide; b) a secondpolypeptide comprising a second class II MHC polypeptide; and c) one ormore immunomodulatory polypeptides, wherein the first and secondpolypeptide form a heterodimer, wherein the first and/or the secondpolypeptide comprises the one or more immunomodulatory polypeptides,wherein the one or more immunomodulatory polypeptides comprises one ormore variant interleukin-2 (IL-2) polypeptides comprising an amino acidsequence having at least 85% amino acid sequence identity to set forthin SEQ ID NO:1, and having one or more amino acid substitutions relativeto set forth in SEQ ID NO:1, and wherein at least one of the one or moreimmunomodulatory polypeptides that is a variant IL-2 polypeptideexhibits reduced binding affinity to an IL-2 receptor (IL2R) comprisingalpha, beta, and gamma polypeptides having amino acid sequences setforth in SEQ ID NOs:54-56, compared to the binding affinity of a controlIL-2 polypeptide comprising the IL-2 amino acid sequence set forth inSEQ ID NO:1 for the IL2R polypeptide.